WO2013050567A1 - Anticorps phosphospécifiques reconnaissant tau - Google Patents

Anticorps phosphospécifiques reconnaissant tau Download PDF

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WO2013050567A1
WO2013050567A1 PCT/EP2012/069783 EP2012069783W WO2013050567A1 WO 2013050567 A1 WO2013050567 A1 WO 2013050567A1 EP 2012069783 W EP2012069783 W EP 2012069783W WO 2013050567 A1 WO2013050567 A1 WO 2013050567A1
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antibody
seq
amino acid
acid sequence
fragment
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PCT/EP2012/069783
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English (en)
Inventor
Andrea Pfeifer
Andreas Muhs
Maria Pihlgren
Oskar Adolfsson
Fred Van Leuven
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Ac Immune S.A.
Katholieke Universiteit Leuven
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Priority claimed from PCT/EP2011/067604 external-priority patent/WO2012045882A2/fr
Priority to BR112014008202-2A priority Critical patent/BR112014008202B1/pt
Priority to JP2014533928A priority patent/JP6358953B2/ja
Priority to EP16180969.4A priority patent/EP3135689B1/fr
Priority to EP12769656.5A priority patent/EP2764022B9/fr
Priority to KR1020147012061A priority patent/KR101981351B1/ko
Priority to ES12769656.5T priority patent/ES2600915T3/es
Priority to MX2014004026A priority patent/MX354662B/es
Application filed by Ac Immune S.A., Katholieke Universiteit Leuven filed Critical Ac Immune S.A.
Priority to CA2850686A priority patent/CA2850686C/fr
Priority to BR122021017560-8A priority patent/BR122021017560B1/pt
Priority to US14/348,918 priority patent/US9540434B2/en
Priority to CN201280060349.3A priority patent/CN104080806B/zh
Priority to RU2014118456A priority patent/RU2639537C2/ru
Publication of WO2013050567A1 publication Critical patent/WO2013050567A1/fr
Priority to HK15100957.4A priority patent/HK1200469A1/xx
Priority to US15/337,964 priority patent/US10066010B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/14—Post-translational modifications [PTMs] in chemical analysis of biological material phosphorylation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention is related to methods and compositions for the therapeutic and diagnostic use in the treatment of diseases and disorders which are caused by or associated with neurofibrillary tangles.
  • the invention relates to antibodies, which specifically recognize and bind to phosphorylated pathological protein tau-conformers and to methods and compositions involving said antibodies for the therapeutic and diagnostic use in the treatment of tauopathies including Alzheimer's Disease (AD).
  • AD Alzheimer's Disease
  • NTs neuropil threads
  • AD Alzheimer's Disease
  • NTs neuropil threads
  • AD neuropil threads
  • They are composed of the microtubule-associated protein tau that has undergone posttranslational modifications, including phosphorylation, deamidation and isomerization on asparaginyl or aspartyl residues. They originate by the aggregation of hyper-phosphorylated protein tau and its conformers. AD shares this pathology with many neurodegenerative tauopathies, in particularly with specified types of frontotemporal dementia (FTD).
  • FTD frontotemporal dementia
  • Protein Tau is a freely soluble, "naturally unfolded" protein that binds avidly to microtubules (MTs) to promote their assembly and stability. Ts are of major importance for the cytoskeletal integrity of neurons - and thereby for the proper formation and functioning of neuronal circuits, hence for learning and memory.
  • the binding of tau to MT is controlled by dynamic phosphorylation and de-phosphorylation, as demonstrated mainly in vitro and in non-neuronal cells. Due to the large number of possible phosphorylation sites (>80), the exact contribution of each and the identity of the responsible kinases remain largely undefined in vivo.
  • AD brain tau pathology develops later than, and therefore probably in response to amyloid pathology, which constitutes the essence of the amyloid cascade hypothesis. This is based on and indicated by studies in AD and Down syndrome patients, and is corroborated by studies in transgenic mice with combined amyloid and tau pathology (Lewis et al., 2001 ; Oddo et al., 2004; Ribe et al., 2005; Muyllaert et al, 2006; 2008; Terwel et al, 2008).
  • tauopathy is not an innocent side-effect but a major pathological executor in AD is based on sound genetic, pathological and experimental observations that corroborate each other fully:
  • the obligate pathogenic cause is amyloid accumulation, but invariably the pathology comprises collateral tauopathy, identical to that in the late- onset sporadic AD cases;
  • AD Alzheimer's disease
  • Immunotherapy targeting tau pathology is anticipated to counteract the pathological protein tau-conformers that are known or postulated to cause synaptic dysfunction and neurodegeneration.
  • the present invention provides for binding proteins recognizing and binding to major pathological phospho-epitopes of the tau protein.
  • the present invention provides specific antibodies against linear and conformational, simple and complex phospho-epitopes on protein tau, particularly on aggregated tau protein that are believed to be responsible for synapto- and neuro-toxicity in tauopathies, including AD.
  • the present invention relates in one embodiment to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, which binding peptide or protein or antibody recognizes and specifically binds to a phospho-epitope on a mammalian, particularly on the human Tau protein or on a fragment thereof, particularly to a phospho-epitope on aggregated Tau protein, particularly to a pathological protein tau conformer, but, in one embodiment, does not bind to the corresponding unphosphorylated epitope and/or to non-related epitopes, wherein said binding peptide or antibody has a high binding affinity to soluble, oligomeric and insoluble phosphorylated Tau protein and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated Tau protein in vivo, particularly in the brain, particularly with a dissociation constant of at least 10 nM, particularly of at least 8 nM.
  • nM particularly of at least 5 nM, particularly of at least 2 nM, particularly of at least 1 nM, particularly of at least 500 pM, particularly of at least 400 pM, particularly of at least 300 pM, particularly of at least 200 pM, particularly of at least 100 pM, particularly of at least 50 pM.
  • the dissociation constant is in a range of between 2 nM and 80 nM, particularly between 2 nM and 40 nM , particularly between 2 nM and 10 nM.
  • an antibody or a functional fragment thereof wherein said antibody or antibody fragment binds to a phospho-epitope having, or within, the amino acid sequence VYKSPWSGDTSPRHL (SEQ ID NO: 62) (Tau aa 393-408 of SEQ ID NO: 67, e.g. , as set forth in Table 1) comprising a phosphorylated Ser at position 396 (pS396) and at position 404 (pS404).
  • the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, which binding peptide or antibody recognizes and specifically binds to a phospho-epitope on a mammalian, particularly on the human Tau protein or on a fragment thereof, particularly to a pathological protein tau conformer, but, in one embodiment, does not bind to the corresponding unphosphorylated epitope and/or to non-related epitopes, wherein said binding peptide or antibody has a high binding affinity to soluble, oligomeric and insoluble phosphorylated Tau protein and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated Tau protein in vivo and has an association rate constant of 10 4 M "1 s "1 or greater, particularly of between 3 - 5 x 10 4 M ' V 1 or
  • the association rate constant is in a range of between 1.6 x 10 3 and 5 x 10 5 , particularly between 2.4 x 10 4 and 5 x 10 5 , between 3 x 10 3 and 5 x 10 5 .
  • the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, which binding peptide or antibody recognizes and specifically binds to a phospho-epitope on a mammalian, particularly on the human Tau protein or on a fragment thereof, particularly to a pathological protein tau conformer, but, in one embodiment, does not bind to the corresponding unphosphorylated epitope and/or to non-related epitopes, wherein said binding peptide or antibody has a high binding affinity to soluble, oligomeric and insoluble phosphorylated Tau protein and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated Tau protein in vivo and has a high binding affinity with a dissociation constant of at least 4 nM and an association rate constant of 10 5 M ' V 1 or greater
  • the dissociation constant is in a range of between 2 nM and 80 nM and the association rate constant is in a range of between 1.6 x 10 3 and 5 x 10 5 , particularly the dissociation constant is in a range of between 2 nM and 40 nM and the association rate constant is in a range of between 2.4 x 10 4 and 5 x 10 5 , particularly the dissociation constant is in a range of between 2 nM and 10 nM and the association rate constant is in a range of between 3 x 10 3 and 5 x 10 5 .
  • One embodiment (4) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, which binding peptide or antibody recognizes and specifically binds to a phospho-epitope on a mammalian, particularly on the human Tau protein or on a fragment thereof, particularly to a pathological protein tau conformer, but, in one embodiment, does not bind to the corresponding unphosphorylated epitope and/or to non-related epitopes, wherein said binding peptide or antibody has a high binding affinity to soluble, oligomeric and insoluble phosphorylated Tau protein and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated Tau protein in vivo and wherein said binding peptide or antibody binds to an epitope on a mammalian, particularly on the human Tau protein as shown in S
  • One embodiment (5) relates to the binding peptide or antibody of any of the preceding embodiments, wherein said peptide binds to an epitope on a mammalian, particularly on the human Tau protein, but especially the human Tau protein as shown in SEQ ID NO: 67, comprising Tau aa 393-401 comprising a phosphorylated Ser at position 396 (pS396).
  • W a mammalian, particularly on the human Tau protein, but especially the human Tau protein as shown in SEQ ID NO: 67, comprising Tau aa 393-401 comprising a phosphorylated Ser at position 396 (pS396).
  • One embodiment (6) relates to the binding peptide or antibody of any o the preceding embodiments, wherein said peptide binds to an epitope on a mammalian, particularly on the human Tau protein, but especially the human Tau protein as shown in SEQ ID NO: 67, comprising Tau aa 396-401 comprising a phosphorylated Ser at position 396 (pS396).
  • One embodiment (7) relates to the binding peptide or antibody of any of the preceding embodiments, wherein said peptide binds to an epitope on a mammalian, particularly on the human Tau protein, but especially the human Tau protein as shown in SEQ ID NO: 67, comprising Tau aa 394-400 comprising a phosphorylated Ser at position 396 (pS396).
  • One embodiment (8) relates to the binding peptide or antibody of any of the preceding embodiments, wherein said peptide binds to an epitope on a mammalian, particularly on the human Tau protein, but especially the human Tau protein as shown in SEQ ID NO: 67, comprising Tau aa 402-406 comprising a phosphorylated Ser at position 404 (pS404).
  • One embodiment (9) relates to the binding peptide or antibody of any of the preceding embodiments, wherein said peptide binds to an epitope on a mammalian, particularly on the human Tau protein, but especially the human Tau protein as shown in SEQ ID NO: 67, comprising Tau aa 393-400 comprising a phosphorylated Ser at position 396 (pS396).
  • One embodiment (10) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, which binding peptide or antibody recognizes and specifically binds to a phospho-epitope on a mammalian, particularly on the human Tau protein or on a fragment thereof, particularly to a pathological protein tau conformer, but, in one embodiment, does not bind to the corresponding unphosphorylated epitope and/or to non-related epitopes wherein said binding peptide or antibody has a high binding affinity to soluble, oligomeric and insoluble phosphorylated Tau protein and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated Tau protein in vivo and wherein said binding peptide or antibody comprises a first binding domain comprising in sequence a CDR1 with the amino acid sequence shown in SEQ ID NO:
  • an antibody or a functional fragment thereof which antibody or fragment thereof recognizes and specifically binds to a phospho-epitope on a mammalian Tau protein or on a fragment thereof, wherein said antibody or fragment thereof comprises:
  • a first binding domain comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 73, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 74, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 75;
  • a second binding domain comprising an amino acid sequence comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 70, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 71 , and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 72.
  • One embodiment (11 ) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, which binding peptide or antibody recognizes and specifically binds to a phospho-epitope on a mammalian, particularly on the human Tau protein or on a fragment thereof, particularly to a pathological protein tau conformer, but, in one embodiment, does not bind to the corresponding unphosphorylated epitope and/or to non-related epitopes wherein said binding peptide or antibody has a high binding affinity to soluble, oligomeric and insoluble phosphorylated Tau protein and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated Tau protein in vivo and wherein said binding peptide or antibody comprises a first binding domain comprising in sequence a CDR1 with the amino acid sequence shown in SEQ ID
  • an antibody or a functional fragment thereof which antibody or fragment thereof recognizes and specifically binds to a phospho-epitope on a mammalian Tau protein or on a fragment thereof, wherein said antibody or fragment thereof comprises:
  • a first binding domain comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 81 , a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 82, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 83;
  • a second binding domain comprising an amino acid sequence comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 78, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 79, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 80.
  • One embodiment (12) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, which binding peptide or antibody recognizes and specifically binds to a phospho-epitope on a mammalian, particularly on the human Tau protein or on a fragment thereof, particularly to a pathological protein tau conformer, but, in one embodiment, does not bind to the corresponding unphosphorylated epitope and/or to non-related epitopes wherein said binding peptide or antibody has a high binding affinity to soluble, oligomeric and insoluble phosphorylated Tau protein and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated Tau protein in vivo and wherein said binding peptide or antibody comprises a first binding domain comprising in sequence a CDR1 with the amino acid sequence shown in SEQ ID NO:
  • an antibody or a functional part thereof which antibody or fragment thereof recognizes and specifically binds to a phospho-epitope on a mammalian Tau protein or on a fragment thereof, wherein said antibody or fragment thereof comprises:
  • a first binding domain comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 93, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 94, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 95;
  • a second binding domain comprising an amino acid sequence comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 89, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 90, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 91.
  • One embodiment (13) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, which binding peptide or antibody recognizes and specifically binds to a phospho-epitope on a mammalian, particularly on the human Tau protein or on a fragment thereof, particularly to a pathological protein tau conformer, but, in one embodiment, does not bind to the corresponding unphosphorylated epitope and/or to non-related epitopes wherein said binding peptide or antibody has a high binding affinity to soluble, oligomeric and insoluble phosphorylated Tau protein and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated Tau protein in vivo and wherein said binding peptide or antibody comprises a first binding domain comprising in sequence a CDR1 with the amino acid sequence shown in SEQ ID NO:
  • an antibody or a functional fragment thereof which antibody or fragment thereof recognizes and specifically binds to a phospho-epitope on a mammalian Tau protein or on a fragment thereof, wherein said antibody or fragment thereof comprises:
  • a first binding domain comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 101 , a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 102, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 103;
  • a second binding domain comprising an amino acid sequence comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 98, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 99, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 100.
  • One embodiment (14) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, which binding peptide or antibody recognizes and specifically binds to a phospho-epitope on a mammalian, particularly on the human Tau protein or on a fragment thereof, particularly to a pathological protein tau conformer, but, in one embodiment, does not bind to the corresponding unphosphorylated epitope and/or to non-related epitopes wherein said binding peptide or antibody has a high binding affinity to soluble, oligomeric and insoluble phosphorylated Tau protein and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated Tau protein in vivo and wherein said binding peptide or antibody comprises a first binding domain comprising in sequence a CDR1 with the amino acid sequence shown in SEQ ID NO:
  • an antibody or a functional fragment thereof which antibody or fragment thereof recognizes and specifically binds to a phospho-epitope on a mammalian Tau protein or on a fragment thereof, wherein said antibody or fragment thereof comprises:
  • a first binding domain comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 106, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 107, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 108;
  • a second binding domain comprising an amino acid sequence comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 89, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 1 15, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 91.
  • the first binding domain of an antibody or antibody fragment thereof described herein is a light chain variable region
  • the second binding domain of an antibody or antibody fragment thereof described herein is a heavy chain variable region
  • the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, which binding peptide or antibody recognizes and specifically binds to a phospho-epitope on a mammalian, particularly on the human Tau protein or on a fragment thereof, particularly to a pathological protein tau conformer, but, in one embodiment, does not bind to the corresponding unphosphorylated epitope and/or to non-related epitopes, wherein said binding peptide or antibody has a high binding affinity to soluble, oligomeric and insoluble phosphorylated Tau protein and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated Tau protein in vivo and wherein said binding peptide or antibody comprises a first binding domain comprising the amino acid sequence shown in SEQ ID NO: 69, 77, 1 16
  • One embodiment (16) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, which binding peptide or antibody recognizes and specifically binds to a phospho-epitope on a mammalian, particularly on the human Tau protein or on a fragment thereof, particularly to a pathological protein tau conformer, but, in one embodiment, does not bind to the corresponding unphosphorylated epitope and/or to non-related epitopes wherein said binding peptide or antibody has a high binding affinity to soluble, oligomeric and insoluble phosphorylated Tau protein and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated Tau protein in vivo and wherein said binding peptide or antibody comprises a first binding domain comprising the amino acid sequence shown in SEQ ID NO: 69, or an amino acid sequence
  • One embodiment (17) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, which binding peptide or antibody recognizes and specifically binds to a phospho-epitope on a mammalian, particularly on the human Tau protein or on a fragment thereof, particularly to a pathological protein tau conformer, but, in one embodiment, does not bind to the corresponding unphosphorylated epitope and/or to non-related epitopes wherein said binding peptide or antibody has a high binding affinity to soluble, oligomeric and insoluble phosphorylated Tau protein and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated Tau protein in vivo and wherein said binding peptide or antibody comprises a first binding domain comprising the amino acid sequence shown in SEQ ID NO: 77, or an amino acid sequence
  • One embodiment (18) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, which binding peptide or antibody recognizes and specifically binds to a phospho-epitope on a mammalian, particularly on the human Tau protein or on a fragment thereof, particularly to a pathological protein tau conformer, but, in one embodiment, does not bind to the corresponding unphosphorylated epitope and/or to non-related epitopes wherein said binding peptide or antibody has a high binding affinity to soluble, oligomeric and insoluble phosphorylated Tau protein and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated Tau protein in vivo and wherein said binding peptide or antibody comprises a first binding domain comprising the amino acid sequence shown in SEQ ID NO: 1 6, 92, or 1 18,
  • One embodiment (19) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, which binding peptide or antibody recognizes and specifically binds to a phospho-epitope on a mammalian, particularly on the human Tau protein or on a fragment thereof, particularly to a pathological protein tau conformer, but, in one embodiment, does not bind to the corresponding unphosphorylated epitope and/or to non-related epitopes wherein said binding peptide or antibody has a high binding affinity to soluble, oligomeric and insoluble phosphorylated Tau protein and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated Tau protein in vivo and wherein said binding peptide or antibody comprises a first binding domain comprising the amino acid sequence shown in SEQ ID NO: 97, or an amino acid sequence
  • One embodiment (20) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, which binding peptide or antibody recognizes and specifically binds to a phospho-epitope on a mammalian, particularly on the human Tau protein or on a fragment thereof, particularly to a pathological protein tau conformer, but, in one embodiment, does not bind to the corresponding unphosphorylated epitope and/or to non-related epitopes wherein said binding peptide or antibody has a high binding affinity to soluble, oligomeric and insoluble phosphorylated Tau protein and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated Tau protein in vivo and wherein said binding peptide or antibody comprises a first binding domain comprising the amino acid sequence shown in SEQ ID NO: 105, or an amino acid sequence
  • One embodiment (21 ) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, wherein said binding peptide or antibody comprises a first binding domain the amino acid sequence shown in SEQ ID NO: 69, or an amino acid sequence at least 99% identical thereto; and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 68, or an amino acid sequence at least 93% identical thereto.
  • One embodiment (22) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, wherein said binding peptide or antibody comprises a first binding domain comprising the amino acid sequence shown in SEQ ID NO: 77, or an amino acid sequence at least 95% identical thereto and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 76, or an amino acid sequence at least 90% identical thereto.
  • One embodiment (23) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, wherein said binding peptide or antibody comprises a first binding domain comprising the amino acid sequence shown in SEQ ID NO: 1 16, 92, or 1 18, or an amino acid sequence at least 93 95% identical thereto; and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 88, or an amino acid sequence at least 93% identical thereto.
  • One embodiment (24) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, wherein said binding peptide or antibody comprises a first binding domain comprising the amino acid sequence shown in SEQ ID NO: 97, or an amino acid sequence at least 99% identical thereto; and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 96, or an amino acid sequence at least 90% identical thereto.
  • One embodiment (25) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, wherein said binding peptide or antibody comprises a first binding domain comprising the amino acid sequence shown in SEQ ID NO: 105, or an amino acid sequence at least 98%, or 99% identical thereto; and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 104, or an amino acid sequence at least 90% identical thereto.
  • One embodiment (26) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, according to embodiment (16), wherein said first binding domain contains the CDRs as shown in SEQ ID NOs: 73-75, and said second binding domain contains the CDRs as shown in SEQ ID NOs: 70-72.
  • One embodiment (27) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, according to embodiment (17), wherein said first binding domain contains the CDRs as shown in SEQ ID NOs: 81-83, and said second binding domain contains the CDRs as shown in SEQ ID NOs: 78-80.
  • One embodiment (28) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, according to embodiment (18), wherein said first binding domain contains the CDRs as shown in SEQ ID NOs: 93-95, and said second binding domain contains the CDRs as shown in SEQ ID NOs: 89-91.
  • One embodiment (29) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, according to embodiment (19), wherein said first binding domain contains the CDRs as shown in SEQ ID NOs: 101-103, and said second binding domain contains the CDRs as shown in SEQ ID NOs: 98-100.
  • One embodiment (30) of the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, according to embodiment (18), wherein said first binding domain contains the CDRs as shown in SEQ ID NOs: 89, 1 15, and 91 , and said second binding domain contains the CDRs as shown in SEQ ID NOs: 106-108.
  • the present invention relates to a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, particularly a binding peptide or antibody of any of the preceding embodiments, which binding peptide or antibody recognizes and specifically binds to a phospho-epitope on a mammalian, particularly on the human Tau protein or on a fragment thereof, particularly to a pathological protein tau conformer, but, in one embodiment, does not bind to the corresponding unphosphorylated epitope and/or to non-related epitopes wherein said binding peptide or antibody has a high binding affinity to soluble, oligomeric and insoluble phosphorylated Tau protein and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated Tau protein in vivo and wherein said binding peptide or antibody comprises a
  • first binding domain comprising the amino acid sequence shown in SEQ ID NO: 69 and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 68; or a
  • first binding domain comprising the amino acid sequence shown in SEQ ID NO: 77 and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 76; or a
  • first binding domain comprising the amino acid sequence shown in SEQ ID NO: 1 16 and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 88; or a;
  • first binding domain comprising the amino acid sequence shown in SEQ ID NO: 92 and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 88; or a
  • first binding domain comprising the amino acid sequence shown in SEQ ID NO: 97 and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 96; or a
  • first binding domain comprising the amino acid sequence shown in SEQ ID NO: 105 and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 104.
  • first binding domain comprising the amino acid sequence shown in SEQ ID NO: 1 18 and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 88
  • the binding peptide of any of the preceding embodiments is an antibody, particularly an antibody of the lgG2a, lgG2b or the lgG3 isotype, particularly a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody or a fully human antibody.
  • One embodiment (33) of the invention relates to a polynucleotide encoding the binding peptide of any one of the preceding embodiments.
  • said polynucleotide comprises a nucleic acid molecule selected from the group consisting of
  • nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence as depicted in SEQ ID NOs: 84-87, SEQ ID NO: 109-1 14, 1 17 and 1 19;
  • nucleic acid molecule comprising a nucleotide sequence that has at least 85% sequence identity to the sequence shown in SEQ ID NOs: 84-87, SEQ ID NO: 109- 114, 1 17 and 1 19;
  • nucleic acid molecule comprising a nucleotide sequence that has at least 90% sequence identity to the sequence shown in SEQ ID NOs: 84-87.
  • nucleic acid molecule comprising a nucleotide sequence that has at least 95% sequence identity to the sequence shown in SEQ ID NOs: 84-87, SEQ ID NO: 109- 1 14, 1 17 and 1 19;
  • nucleic acid molecule comprising a nucleotide sequence that has at least 98% sequence identity to the sequence shown in SEQ ID NOs: 84-87, SEQ ID NO: 109- 1 14, 1 17 and 1 19;
  • nucleic acid molecule comprising a nucleotide sequence that has at least 99% sequence identity to the sequence shown in SEQ ID NOs: 84-87, SEQ ID NO: 109- 1 14, 1 17 and 1 19;
  • nucleic acid molecule comprising a nucleotide sequence the complementary strand of which hybridizes to the nucleic acid molecule of any of a) - f);
  • nucleic acid molecule comprising a nucleotide sequence that deviates from the nucleotide sequence defined in any of a) - g) by the degeneracy of the genetic code, wherein said nucleic acid molecule as defined in any of a) - h) recognizes and specifically binds to a phospho-epitope on a mammalian, particularly on the human Tau protein or on a fragment thereof, particularly on the human Tau protein as shown in SEQ ID NO: 67, selected from the group consisting of Tau aa 393-401 , comprising a phosphoryiated Ser at position 396 (pS396), Tau aa 396-401 comprising a phosphoryiated Ser at position 396 (pS396), Tau aa 394-400 comprising a phosphoryiated Ser at position 396 (pS396), Tau aa 402-406 comprising a phosphorylated Ser at position 404 (pS404), and Tau aaa 393-
  • a binding peptide is provided or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide, according to any one of the preceding embodiments, or a combination thereof, which is capable of specifically recognizing and binding to a phospho- epitope on a mammalian, particularly on the human Tau protein, particularly a microtubule- associated protein tau, particularly an aggregated microtubule-associated and hyperphosphorylated protein tau such as that present in paired helical filaments (PHF), which are the predominant structures in neurofibrillary tangles, neuropil threads and dystrophic neurites. but, in one embodiment, does not bind to the corresponding unphosphorylated epitope and/or to non-related epitopes.
  • PHF paired helical filaments
  • the human tau protein is the human Tau protein as shown in SEQ !D NO; 67.
  • binding peptides and antibodies according to any one of the preceding embodiments can thus be used (37) for reducing the levels of total soluble tau protein, particularly of soluble phosphorylated tau protein, in the brain, particularly in the brain cortex and/or hippocampus, of a mammal or a human containing increased levels of soluble tau protein and/or soluble phosphorylated tau protein.
  • binding peptides and antibodies according to any one of the preceding embodiments can also be used (38) for reducing the levels of paired helical filaments containing hyperphosphorylated tau protein (pTau PHF) in the brain, particularly in the brain cortex and/or hippocampus, of a mammal or a human containing increased levels of said pTau paired helical filaments.
  • pTau PHF hyperphosphorylated tau protein
  • binding peptides and antibodies according to any one of the preceding embodiments can therefore be used (40) in therapy, particularly in human therapy, for slowing or halting the progression of a tau-protein-associated disease, disorder or condition.
  • binding peptides and antibodies according to any one of the preceding embodiments can further be used (41 ) in therapy, particularly in human therapy, for improving or alleviating the symptoms associated with tau-protein-associated diseases, disorders or conditions such as, for example, impairment or loss of cognitive functions including reasoning, situational judgement, memory capacity, learning, special navigation, etc.
  • the invention relates to the binding peptides and antibodies according to any one of the preceding embodiments for use in therapy, particularly for use in the treatment of tauopathies, a group of tau-protein-associated diseases and disorders, or for alleviating the symptoms associated with tauopathies.
  • the invention relates to the binding peptides and antibodies according to any one of the preceding embodiments for retaining or increasing cognitive memory capacity in a mammal suffering from a tauopathy.
  • binding peptides and antibodies comprising at least one or all of the light chain CDRs of antibodies ACI-35-2A1 -Ab1 ; ACI-35- 2A1-Ab2; ACI-35-4A6-Ab1 ; ACI-35-4A6-Ab2; ACI-35-1 D2-Ab1 ; ACI-35-2G5-Ab1 ; ACI-35- 2G5-Ab2; ACI-35-2G5-Ab3: as given in SEQ ID NOs: 73-75, 81 -83, 93-95, 101-103, 106-108 and/or at least one or all of the heavy chain CDRs of antibodies ACI-35-2A1 -Ab1 ; ACI-35- 2A1 -Ab2; ACI-35-4A6-Ab1 ; ACI-35-4A6-Ab2; ACI-35-1 D2-Ab1 ; ACI-35-2G5-Ab1 ; ACI-35- 2G5-Ab2
  • binding peptides and antibodies comprising at least one or all of the light chain CDRs of antibodies ACI-35-2G5-Ab2; ACI-35- 2G5-Ab3 as given in SEQ ID NOs: 106-108 and/or at least one or all of the heavy chain CDRs of antibodies ACI-35-2G5-Ab2; ACI-35-2G5-Ab3; as given in SEQ ID NOs: 89, 1 15 and 91 , are used in therapy, particularly in human therapy, for improving or alleviating the symptoms associated with tau-protein-associated diseases, disorders or conditions such as, for example, impairment or loss of cognitive functions including reasoning, situational judgement, memory, learning, special navigation, etc.
  • Binding of the peptides or antibodies according to the preceding embodiments to tau tangles and pTau on brains may be determined by applying protein immuno-reactivity testing of selected brain sections and by Western blotting of brain homogenates, respectively, as described in the Examples.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a binding peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide, according to any one of the preceding embodiments, or a combination thereof, in a therapeutically effective amount together with a pharmaceutically acceptable carrier.
  • the binding peptide or a functional part thereof is used in therapy, particularly in human therapy for the treatment or alleviation of the symptoms of tau-protein-associated diseases or disorders including neurodegenerative disorders such as tauopathies.
  • binding peptides, antibodies and/or pharmaceutical compositions may thus be used (48) for slowing or halting the progression of a tau-protein-associated disease, disorder or condition, upon administration of said binding peptides, antibodies and/or pharmaceutical compositions to an animal, particularly a mammal, particularly a human, suffering from such a disease or condition.
  • binding peptides, antibodies and/or pharmaceutical compositions may further be used (49) for improving or alleviating the symptoms associated with tau-protein-associated diseases, disorders or conditions such as, for example, impairment or loss of cognitive functions including reasoning, situational judgement, memory capacity, learning, special navigation, etc, upon administration of said binding peptides, antibodies and/or pharmaceutical compositions to an animal, particularly a mammal, particularly a human, suffering from such a disease or condition.
  • the binding peptide or a functional part thereof is used in the treatment of diseases and disorders which are caused by or associated with the formation of neurofibrillary lesions, the predominant brain pathology in tauopathy comprising a heterogeneous group of neurodegenerative diseases or disorders including diseases or disorders which manifest both tau and amyloid pathologies including, but not limited to, Alzheimer's Disease, Creutzfeldt-Jacob disease, Dementia pugilistica, Down's Syndrome, Gerstmann-Straussier-Scheinker disease, inclusion-body myositis, and prion protein cerebral amyloid angiopathy, traumatic brain injury and further diseases or disorders which do not show a distinct amyloid pathology including, but not limited to, amyotrophic lateral sclerosis/parkinso
  • the binding peptide or a functional part thereof is used in the treatment of Alzheimer ' s Disease.
  • modulation relates to reducing the levels of soluble tau protein, particularly of soluble phosphorylated tau protein, in the brain, particularly in the brain cortex and/or hippocampus, of an animal, particularly a mammal or a human containing increased levels of soluble tau protein and/or soluble phosphorylated tau protein.
  • a method for reducing the levels of insoluble tau protein, particularly of paired helical filaments containing hyperphosphorylated tau protein (pTau PHF) in the brain, particularly in the brain cortex and/or hippocampus, of an animal, particularly a mammal or a human, containing increased levels of insoluble tau protein, particularly of pTau paired helical filaments (pTau PHF) comprising administering to said animal, particularly to said mammal or human, the binding peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide or a pharmaceutical composition, according to any one of the preceding embodiments, or a combination thereof.
  • the present invention relates to a method for slowing or halting the progression of a tau-protein-associated disease, disorder or condition in an animal, particularly a mammal or human comprising administering to said animal, particularly said mammal or human, suffering from such a disease or condition, the binding peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide or a pharmaceutical composition, according to any one of the preceding embodiments, or a combination thereof.
  • the present invention relates to a method for improving or alleviating the symptoms associated with tau-protein-associated diseases, disorders or conditions such as, for example, impairment or loss of cognitive functions including reasoning, situational judgement, memory capacity, learning, special navigation, etc., in an animal, particularly a mammal or a human, comprising administering to said animal, particularly to said mammal or human, suffering from such a disease or condition, the binding peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide or a pharmaceutical composition, according to any one of the preceding embodiments, or a combination thereof.
  • the present invention relates to a method for retaining or increasing cognitive memory capacity in a mammal suffering from a tauopathy.
  • a method for the treatment of a tau-protein-associated disease or disorder including a neurodegenerative disease or disorder such as a tauopathy comprising administering to an animal, particularly to a mammal, but especially to human, suffering from such a disease or disorder, the binding peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide or a pharmaceutical composition, according to any one of the preceding embodiments, or a combination thereof.
  • a method for the treatment of diseases and disorders which are caused by or associated with the formation of neurofibrillary lesions, the predominant brain pathology in tauopathy comprising a heterogeneous group of neurodegenerative diseases or disorders including diseases or disorders which manifest both tau and amyloid pathologies including, but not limited to.
  • a method for inducing a passive immune response in an animal, particularly a mammal or a human, suffering from a neurodegenerative disorder such as tauopathy by administering to said animal or human the binding peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide, or a pharmaceutical composition, according to any one of the preceding embodiments, or a combination thereof.
  • a method of diagnosing a tau-protein- associated disease, disorder or condition in a patient comprising detecting the immunospecific binding of a binding peptide or an active fragment thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, according to any one of the preceding embodiments, to an epitope of the tau protein in a sample or in situ which includes the steps of
  • a binding peptide or a fragment thereof particularly an antibody, particularly a monoclonal antibody or a functional part thereof, according to any one of the preceding claims, wherein said binding peptide or antibody or fragment thereof binds an epitope of the tau protein;
  • binding peptide particularly said antibody, particularly said monoclonal antibody or a functional part thereof, to bind to the tau protein to form an immunological complex
  • a method for diagnosing a predisposition to tau-protein-associated disease, disorder or condition in a patient comprising detecting the immunospecific binding of a binding peptide or an active fragment thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, according to any one of the preceding embodiments, to an epitope of the tau protein in a sample or in situ, which includes the steps of
  • a binding peptide or an active fragment thereof particularly an antibody, particularly a monoclonal antibody or a functional part thereof, according to any one of the preceding embodiments, which peptide or fragment thereof binds an epitope of the tau protein
  • binding peptide particularly said antibody, particularly said monoclonal antibody or a functional part thereof, to bind to the tau antigen to form an immunological complex
  • an increase in the amount of said aggregate compared to a normal control value indicates that said patient is suffering from or is at risk of developing an tau- protein- associated disease or condition.
  • binding peptide particularly said antibody, particularly said monoclonal antibody or a functional part thereof, to bind to the tau antigen to form an immunological complex
  • a binding peptide or an active fragment thereof particularly an antibody, particularly a monoclonal antibody or a functional part thereof according to any one of the preceding embodiments, which peptide or fragment thereof binds to an epitope of the tau protein
  • binding peptide particularly said antibody, particularly said monoclonal antibody or a functional part thereof, to bind to the tau antigen to form an immunological complex
  • Anti-Tau antibodies and fragments thereof may be used in the above methods of the invention.
  • the sample containing the antibody or fragment thereof may be immune-enriched to increase the concentration of Tau protein in the sample by contacting the sample with an anti-Tau antibody or a fragment thereof attached to a solid support.
  • the sample Prior to the step step (a), the sample is immune-enriched to increase the concentration of Tau protein in the sample by contacting the sample with an anti-Tau antibody or a fragment thereof attached to a solid support
  • the invention in another embodiment (64), relates to a test kit for detection and diagnosis of tau-protein-associated diseases, disorders or conditions comprising a binding peptide or an active fragment thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, according to any one of the preceding embodiments.
  • said test kit comprises a container holding one or more binding peptides or active fragments thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, according to any one of the preceding embodiments and instructions for using the binding peptides or antibodies for the purpose of binding to tau antigen to form an immunological complex and detecting the formation of the immunological complex such that presence or absence of the immunological complex correlates with presence or absence of tau antigen.
  • the present invention relates to an epitope on a mammalian, particularly on the human Tau protein as shown in SEQ ID NO: 67, selected from the group consisting of Tau aa 393-401 , comprising a phosphorylated Ser at position 396 (pS396), Tau aa 396-401 comprising a phosphorylated Ser at position 396 (pS396), Tau aa 394-400 comprising a phosphorylated Ser at position 396 (pS396), Tau aa 402-406 comprising a phosphorylated Ser at position 404 (pS404) and Tau aa 393-400 comprising a phosphorylated Ser at position 396 (pS396).
  • said epitope consists of Tau aa 393-401 , comprising a phosphorylated Ser at position 396 (pS396).
  • said epitope consists of Tau aa 396-401 comprising a phosphorylated Ser at position 396 (pS396).
  • said epitope consists of Tau aa 394-400 comprising a phosphorylated Ser at position 396 (pS396).
  • said epitope consists of Tau aa 402-406 comprising a phosphorylated Ser at position 404 (pS404).
  • said epitope consists of Tau aa 393-400 comprising a phosphorylated Ser at position 396 (pS396).
  • the invention in another embodiment (72), relates to a cell line producing a binding peptide or an active fragment thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof according to any one of the preceding embodiments.
  • the invention relates to a cell line, which is hybridoma cell line A4- 4A6-48 deposited on August 30, 201 1 as DSM ACC3 36.
  • the invention relates to a cell line, which is hybridoma cell line A6- 2G5-30 deposited on August 30, 201 1 as DSM ACC3137.
  • the invention relates to a cell line, which is hybridoma cell line A6- 2G5-41 deposited on August 30, 2011 as DSM ACC3138.
  • the invention relates to a cell line, which is hybridoma cell line A4- 2A1-18 deposited on August 30, 201 1 as DSM ACC3139.
  • the invention relates to a cell line, which is hybridoma cell line A4- 2A -40 deposited on August 30, 201 1 as DSM ACC3140. In one embodiment (78), the invention relates to a cell line, which is hybridoma cell line A6- 1 D2-12 deposited on September 6, 201 1 as DSM ACC3141.
  • the invention relates to a monoclonal antibody or a functional part thereof comprising a light chain (VL) and/or a heavy chain (VH) domain, which is encoded by a polynucleotide located on a nucleotide fragment that can be obtained by PCR amplification of DNA of hybridoma cell line A4-2A1-18 deposited on August 30, 201 as DSM ACC3139 using
  • a primer pair comprising a 5'-primer of SEQ ID NO: 149 and a 3'-primer of SEQ ID NO:
  • a mix of primers comprising a 5'-primer selected from the group consisting of SEQ !D NOs: 120, 123, 124, 136, 137, 138, 139, and 140 and a 3'-primer selected from the group consisting of SEQ ID NOs: 131 , 134, and 141-148, for amplification of a second binding domain.
  • the invention relates to a monoclonal antibody or a functional part thereof comprising a light chain (VL) and/or a heavy chain (VH) domain, which is encoded by a polynucleotide located on a nucleotide fragment that can be obtained by PCR amplification of DNA of hybridoma cell line A6-2G5-30 deposited on August 30, 201 1 as DSM ACC3137 using
  • a mix of primers comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 51 and 169-174 and a 3'-primer of SEQ ID NO: 51 , for amplification of a first binding domain; and/or
  • a mix of primers comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 124, 127, and 150-158 and a 3'-primer selected from the group consisting of SEQ ID NOs: 130, and 159-168, for amplification of a second binding domain.
  • the invention relates to a monoclonal antibody or a functional part thereof comprising a light chain (VL) and/or a heavy chain (VH) domain, which is encoded by a polynucleotide located on a nucleotide fragment that can be obtained by PCR amplification of DNA of hybridoma cell line A4-2A1 -40 deposited on August 30, 201 1 as DSM ACC3140 using
  • a mix of primers comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 178, 179 and 180 and a 3'-primer of SEQ ID NO: 51 , for amplification of a first binding domain; and/or
  • a mix of primers comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 121 , 127, 139, 154, 155, and 75 and a 3'-primer selected from the group consisting of SEQ ID NOs: 128, 129, 147, 176, and 177, for amplification of a second binding domain.
  • the invention relates to a monoclonal antibody or a functional part thereof comprising a light chain (VL) and/or a heavy chain (VH) domain, which is encoded by a polynucleotide located on a nucleotide fragment that can be obtained by PGR amplification of DNA of hybridoma cell line A6-2G5-41 deposited on August 30, 20 1 as DSM ACC3138 using
  • a mix of primers comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 51 and 188-192 and a 3'-primer of SEQ ID NO: 51 , for amplification of a first binding domain; and/or
  • a mix of primers comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 120, 124, 126, 181 , 182 and 183 and a 3'-primer selected from the group consisting of SEQ ID NOs: 144, 145 and 184-187, for amplification of a second binding domain.
  • the invention relates to a monoclonal antibody or a functional part thereof comprising a light chain (VL) and/or a heavy chain (VH) domain, which is encoded by a polynucleotide located on a nucleotide fragment that can be obtained by PGR amplification of DNA of hybridoma cell line A4-4A6-48 deposited on August 30, 201 1 as DSM ACC3136 using
  • a mix of primers comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 50 and 201-204 and a 3'-primer of SEQ ID NO: 51 , for amplification of a first binding domain; and/or
  • a mix of primers comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 121 , 137, 151 and 193-197 and a 3'-primer selected from the group consisting of SEQ ID NOs: 131 , 141 , 144, 166, 198, 99 and 200, for amplification of a second binding domain.
  • the invention relates to a monoclonal antibody or a functional part thereof comprising a light chain (VL) and/or a heavy chain (VH) domain, which is encoded by a polynucleotide located on a nucleotide fragment that can be obtained by PCR amplification of DNA of hybridoma cell line A6-1 D2-12 deposited on September 6, 201 1 as DSM ACC3141 using
  • a mix of primers comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 209-214, and 219-221 a 3'-primer of SEQ ID NO: 215, for amplification of a first binding domain; and/or b. a mix of primers comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 216, 217 and 218 and a 3'-primer of SEQ ID NOs: 208, for amplification of a second binding domain.
  • the antibody according to any one of the preceding embodiments may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a camelid antibody, a diabody, or a modified or engineered antibody.
  • the invention provides a method for producing the binding peptides or antibodies of any one of the preceding embodiments, comprising the step of culturing the cell line of any of the preceding embodiments in a suitable cultivation medium and, optionally, purifying the binding peptides or antibody from the cell line or cultivation medium.
  • the present invention provides a method of detecting phosphoTau (pTau) multimers in a brain sample comprising
  • the invention relates in a specific embodiment (88) to a method of post mortem detection of phosphoTau (pTau) multimers in brain homogenates from a subject suspected to suffer from a tau-associated disease or disorder and from a healthy control subject comprising
  • an increase in the amount or intensity of said immunological complex compared to the control value indicates that said patient had suffered from a minimal residual disease.
  • the increase observed in the test sample compared to the control sample is between 30% and 50%, particularly between 35% and 45%.
  • the invention provides an a binding peptide or protein or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, according to any one of the preceding embodiment, which antibody or fragment shows a favourable pK profile.
  • said antibody or fragment has a hight serum concentration up to 10-day post administration, which indicates a pharmacokinetic (PK) profile that favorably supports the use of said antibodies as a therapeutic antibody.
  • Figure 1 shows results of detection of phosphorylated Tau multimers by ACI-35-2A1-Ab1 (left panel), ACI-35-2G5-Ab3 (middle panel), and a control antibody (HT7: right panel) in human brain homogenates from control and AD subjects.
  • Figure 2 shows results detection of total and p-Tau by commercial antibodies in human brain homogenates.
  • Figure 3 shows Detection of phospho-Tau by ACI-35-2A1-A (A), ACI-35-1 D2- Ab1 (B), and ACI-35-2G5-Ab3 (C) in human brain homogenates.
  • Figure 4 shows results of detection of Tau-pS396 in human AD and control (Ctrl) brain by ACI-35-2A1 -Ab1 (A), ACI-35-1 D2-Ab1 (B), and ACI-35-2G5-Ab3 (C) antibodies using AlphaLISA. ****p ⁇ 0.0001 , **p ⁇ 0.01 by Mann-Whitney test.
  • Figure 5 shows results of Tau-pT231 (AT 180) IHC staining in the amygdala (A) and hippocampus (B), following treatment of Tau transgenic mice with ACI-35-2G5-Ab3.
  • Figure 6 shows results of total Tau (HT7) IHC staining in the amygdala (A) and hippocampus (B) , following treatment of Tau transgenic mice with ACI-35-2G5-Ab3.
  • Figure 7 shows an SDS-PAGE for Tau-pS396 generated using different GSK3 conditions, and the membrane blotted using the ACI-35-2G5-Ab3 antibody.
  • Figure 8 shows specific AlphaLISA assay setup using ACI-35-2G5-Ab3-BT and Tau- 3 antibodies.
  • Figure 9 shows detection of Tau-pS396 in human S1 brain fraction from one AD donor; comparison of signal obtained from samples enriched by IP and non-IP samples.
  • SEQ ID NO: 46 - 57 depicts the nucleotide sequences of VH/VK forward and reverse primers.
  • SEQ ID NO: 62 depicts the amino acid sequence of Tau antigen, peptide T3 (see Table 1 ).
  • SEQ ID NO: 67 depicts the amino acid sequence of longest isoform of human tau (441 aa) also called Tau40.
  • SEQ ID NO: 68 depicts the amino acid sequence of the heavy chain variable region (VH) of monoclonal antibody ACI-35-4A6-Ab1 produced by hybridoma cell line A4-4A6-18.
  • SEQ ID NO: 69 depicts the amino acid sequence of the light chain variable region (VK) of monoclonal antibody ACI-35-4A6-Ab1 produced by hybridoma cell line A4-4A6-18.
  • SEQ ID NO: 70 depicts the amino acid sequence of the CDR1 of the heavy chain variable region (VH) of monoclonal antibody ACI-35-4A6-Ab1.
  • SEQ ID NO: 71 depicts the amino acid sequence of the CDR2 of the heavy chain variable region (VH) of monoclonal antibody ACI-35-4A6-Ab1.
  • SEQ ID NO: 72 depicts the amino acid sequence of the CDR3 of the heavy chain variable region (VH) of monoclonal antibody ACI-35-4A6-Ab1.
  • SEQ ID NO: 73 depicts the amino acid sequence of the CDR1 of the light chain variable region (VK) of monoclonal antibody ACI-35-4A6-Ab1 .
  • SEQ ID NO: 74 depicts the amino acid sequence of the CDR2 of the light chain variable region (VK) of monoclonal antibody ACI-35-4A6-Ab .
  • SEQ ID NO: 75 depicts the amino acid sequence of the CDR3 of the light chain variable region (VK) of monoclonal antibody ACI-35-4A6-Ab1.
  • SEQ ID NO: 76 depicts the amino acid sequence of the heavy chain variable region (VH) of monoclonal antibody ACI-35-1 D2-Ab1 produced by hybridoma cell line A6-1 D2-12.
  • SEQ ID NO: 77 depicts the amino acid sequence of the light chain variable region (VK) of monoclonal antibody ACI-35-1 D2-Ab1 produced by hybridoma cell line A6-1 D2-12.
  • SEQ ID NO: 78 depicts the amino acid sequence of the CDR1 of the heavy chain variable region (VH) of monoclonal antibody ACI-35-1 D2-Ab1.
  • SEQ ID NO: 79 depicts the amino acid sequence of the CDR2 of the heavy chain variable region (VH) of monoclonal antibody ACI-35-1 D2-Ab1 .
  • SEQ ID NO: 80 depicts the amino acid sequence of the CDR3 of the heavy chain variable region (VH) of monoclonal antibody ACI-35-1 D2-Ab1.
  • SEQ ID NO: 81 depicts the amino acid sequence of the CDR1 of the light chain variable region (VK) of monoclonal antibody ACI-35-1 D2-A .
  • SEQ ID NO: 82 depicts the amino acid sequence of the CDR2 of the light chain variable region (VK) of monoclonal antibody ACI-35-1 D2-Ab1.
  • SEQ ID NO: 83 depicts the amino acid sequence of the CDR3 of the light chain variable region (VK) of monoclonal antibody ACI-35-1 D2-Ab1 .
  • SEQ ID NO: 84 depicts the nucleotide sequence of the heavy chain variable region (VH) of monoclonal antibody ACI-35-4A6-Ab1 produced by hybridoma cell line A4-4A6-18.
  • SEQ ID NO: 85 depicts the nucleotide sequence of the light chain variable region (VK) of monoclonal antibody ACl-35-4A6-Ab1 produced by hybridoma cell line A4-4A6-18.
  • SEQ ID NO: 86 depicts the nucleotide sequence of the heavy chain variable region (VH) of monoclonal antibody ACI-35-1 D2-Ab1 produced by hybridoma cell line A6-1 D2-12.
  • SEQ ID NO: 87 depicts the nucleotide sequence of the light chain variable region (VK) of monoclonal antibody ACI-35-1 D2-Ab1 produced by hybridoma cell line A6- D2-12.
  • SEQ ID NO: 88 depicts the amino acid sequence of the heavy chain variable region (VH) of monoclonal antibody ACI-35-2A1 -Ab1 , ACI-35-2A1-Ab2, and ACI-35-4A6-Ab2, respectively, produced by hybridoma cell line A4-2A1 -18, A4-2A1 -40 and A4-4A6-48, respectively.
  • VH heavy chain variable region
  • SEQ ID NO: 89 depicts the amino acid sequence of the CDR1 of the heavy chain variable region (VH) of monoclonal antibody ACI-35-2A1-Ab1 , ACI-35-2A1-Ab2, ACI-35-4A6-Ab2, ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively.
  • SEQ ID NO: 90 depicts the amino acid sequence of the CDR2 of the heavy chain variable region (VH) of monoclonal antibody ACI-35-2A1 -Ab1 , ACI-35-2A1 -Ab2, and ACI-35-4A6- Ab2, respectively.
  • SEQ ID NO: 91 depicts the amino acid sequence of the CDR3 of the heavy chain variable region (VH) of monoclonal antibody ACI-35-2A1 -Ab1 , ACI-35-2A1 -Ab2, ACI-35-4A6-Ab2.
  • SEQ ID NO: 92 depicts the amino acid sequence of the light chain variable region (VK) of monoclonal antibody ACI-35-2A1-Ab2 produced by hybridoma cell line A4-2A1 -40
  • SEQ ID NO: 93 depicts the amino acid sequence of the CDR1 of the light chain variable region (VK) of monoclonal antibody ACI-35-2A1-Ab2.
  • SEQ ID NO: 94 depicts the amino acid sequence of the CDR2 of the light chain variable region (VK) of monoclonal antibody ACI-35-2A1-Ab2.
  • SEQ ID NO: 95 depicts the amino acid sequence of the CDR3 of the light chain variable region (VK) of monoclonal antibody ACI-35-2A1-Ab2.
  • SEQ ID NO: 96 depicts the amino acid sequence of the heavy chain variable region (VH) of monoclonal antibody ACI-35-2G5-Ab1 produced by hybridoma cell line A6-2G5-08.
  • SEQ ID NO: 97 depicts the amino acid sequence of the light chain variable region (VK) of monoclonal antibody ACI-35-2G5-Ab1 produced by hybridoma cell line A6-2G5-08.
  • SEQ ID NO: 98 depicts the amino acid sequence of the CDR1 of the heavy chain variable region (VH) of monoclonal antibody ACI-35-2G5-Ab1 .
  • SEQ ID NO: 99 depicts the amino acid sequence of the CDR2 of the heavy chain variable region (VH) of monoclonal antibody ACI-35-2G5-Ab1.
  • SEQ ID NO: 100 depicts the amino acid sequence of the CDR3 of the heavy chain variable region (VH) of monoclonal antibody ACI-35-2G5-Ab1.
  • SEQ ID NO: 101 depicts the amino acid sequence of the CDR1 of the light chain variable region (VK) of monoclonal antibody ACi-35-2G5-Ab1.
  • SEQ ID NO: 102 depicts the amino acid sequence of the CDR2 of the light chain variable region (VK) of monoclonal antibody ACI-35-2G5-Ab1.
  • SEQ ID NO: 103 depicts the amino acid sequence of the CDR3 of the light chain variable region (VK) of monoclonal antibody ACI-35-2G5-A .
  • SEQ ID NO: 104 depicts the amino acid sequence of the heavy chain variable region (VH) of monoclonal antibody ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively, produced by hybridoma cell line A6-2G5-30 and A6-2G5-41 , respectively.
  • VH heavy chain variable region
  • SEQ ID NO: 105 depicts the amino acid sequence of the light chain variable region (VK) of monoclonal antibody ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively, produced by hybridoma cell line A6-2G5-30 and A6-2G5-41 , respectively.
  • VK light chain variable region
  • SEQ ID NO: 106 depicts the amino acid sequence of the CDR1 of the light chain variable region (VK) of monoclonal antibody ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively.
  • SEQ ID NO: 107 depicts the amino acid sequence of the CDR2 of the light chain variable region (VK) of monoclonal antibody ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively.
  • SEQ ID NO: 108 depicts the amino acid sequence of the CDR3 of the light chain variable region (VK) of monoclonal antibody ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively.
  • SEQ ID NO: 109 depicts the nucleotide sequence of the heavy chain variable region (VH) of monoclonal antibody ACI-35-2A1 -Ab1 , ACI-35-2A1-Ab2, and ACI-35-4A6-Ab2, respectively, produced by hybridoma cell line A4-2A1-18, A4-2A1-40 and A4-4A6-48, respectively.
  • VH heavy chain variable region
  • SEQ ID NO: 1 10 depicts the nucleotide sequence of the light chain variable region (VK) of monoclonal antibody ACI-35-2A1 -Ab2 produced by hybridoma cell line A4-2A1-40.
  • SEQ ID NO: 1 11 depicts the nucleotide sequence of the heavy chain variable region (VH) of monoclonal antibody ACI-35-2G5-AB1 produced by hybridoma ceil line A6-2G5-08.
  • SEQ ID NO: 1 12 depicts the nucleotide sequence of the light chain variable region (VK) of monoclonal antibody ACI-35-2G5-AB1 produced by hybridoma cell line A6-2G5-08.
  • SEQ ID NO: 13 depicts the nucleotide sequence of the heavy chain variable region (VH) of monoclonal antibody ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively, produced by hybridoma cell line A6-2G5-30 and A6-2G5-41 , respectively.
  • VH heavy chain variable region
  • SEQ ID NO: 1 14 depicts the nucleotide sequence of the light chain variable region (VK) of monoclonal antibody ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively, produced by hybridoma cell line A6-2G5-30 and A6-2G5-4 , respectively.
  • VK light chain variable region
  • SEQ ID NO: 1 15 depicts the amino acid sequence of the CDR2 of the heavy chain variable region (VH) of monoclonal antibody ACI-35-2G5-AB2 and ACI-35-2G5-AB3.
  • SEQ ID NO: 1 16 depicts the amino acid sequence of the light chain variable region (VK) of monoclonal antibody ACI-35-2A1 -Ab1 produced by hybridoma cell line A4-2A1 -18.
  • SEQ ID NO: 1 17 depicts the nucleotide sequence of the light chain variable region (VK) of monoclonal antibody ACI-35-2A1-Ab1 produced by hybridoma cell line A4-2A1 -18.
  • SEQ ID NO: 1 18 depicts the amino acid sequence of the light chain variable region (VK) of monoclonal antibody ACI-35-4A6-Ab2 produced by hybridoma cell line A4-4A6-48.
  • SEQ ID NO: 1 19 depicts the nucleotide sequence of the light chain variable region (VK) of monoclonal antibody ACI-35-4A6-Ab2 produced by hybridoma cell line A4-4A6-48.
  • SEQ ID NO: 120 - 221 depicts the nucleotide sequences of VH VK forward and reverse primers.
  • polypeptide polypeptide
  • peptide protein
  • binding peptide are used herein interchangeably and refer to chains of amino acids (typically L-amino acids) whose alpha carbons are linked through peptide bonds formed by a condensation reaction between the carboxyl group of the alpha carbon of one amino acid and the amino group of the alpha carbon of another amino acid.
  • the terminal amino acid at one end of the chain i.e., the amino terminal
  • the terminal amino acid at the other end of the chain i.e., the carboxy terminal
  • amino terminus refers to the free alpha-amino group on the amino acid at the amino terminal of the peptide, or to the alpha-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide.
  • carboxy terminus refers to the free carboxyl group on the amino acid at the carboxy terminus of a peptide, or to the carboxyl group of an amino acid at any other location within the peptide.
  • a binding peptide may constitutes antibodies such as polyclonal or monoclonal antibodies, human or humanized antibodies, diabodies, camelid antibodies, etc, or functional parts thereof as defined herein.
  • fragment thereof or “fragment” as used herein in the context of a peptide refer to a functional peptide fragment which has essentially the same (biological) activity as an intact peptide defined herein.
  • the terms when used herein in the context of an antibody refers to an antibody fragment comprising a portion of an intact antibody that contains an antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; diabodies: single-chain antibody molecules, including single-chain Fv (scFv) molecules; and bispecific and multispecific antibodies and/or antibody fragments.
  • amino acids making up a peptide are numbered in order, starting at the amino terminal and increasing in the direction toward the carboxy terminal of the peptide.
  • that amino acid is positioned closer to the carboxy terminal of the peptide than the preceding amino acid.
  • amino acid is used herein to refer to an amino acid that is incorporated into a peptide by an amide bond.
  • the amino acid may be a naturally occurring amino acid or, unless otherwise limited, may encompass known analogs of natural amino acids that function in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics).
  • an amide bond mimetic includes peptide backbone modifications well known to those skilled in the art.
  • the phrases "isolated” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany it as found in its native state. Thus, the peptides described herein do not contain materials normally associated with their in situ environment. Typically, the isolated, immunogenic peptides described herein are at least about 80% pure, usually at least about 90%, and preferably at least about 95% as measured by band intensity on a silver stained gel.
  • Protein purity or homogeneity may be indicated by a number of methods well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization upon staining. For certain purposes high resolution will be needed and HPLC or a similar means for purification utilized.
  • the immunogenic peptides are relatively short in length (i.e., less than about 50 amino acids), they are often synthesized using standard chemical peptide synthesis techniques.
  • Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is a preferred method for the chemical synthesis of the immunogenic peptides described herein. Techniques for solid phase synthesis are known to those skilled in the art.
  • the immunogenic peptides described herein are synthesized using recombinant nucleic acid methodology. Generally, this involves creating a nucleic acid sequence that encodes the peptide, placing the nucleic acid in an expression cassette under the control of a particular promoter, expressing the peptide in a host, isolating the expressed peptide or polypeptide and, if required, renaturing the peptide. Techniques sufficient to guide one of skill through such procedures are found in the literature.
  • recombinant peptides can be purified according to standard procedures, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Substantially pure compositions of about 50 % to 95 % homogeneity are preferred, and 80 % to 95 % or greater homogeneity is most preferred for use as therapeutic agents.
  • the immunogenic peptides may possess a conformation substantially different than the native conformations of the constituent peptides. In this case, it is often necessary to denature and reduce the antiproliferative peptide and then to cause the peptide to re-fold into the preferred conformation. Methods of reducing and denaturing proteins and inducing refolding are well known to those of skill in the art.
  • Antigenicity of the purified protein may be confirmed, for example, by demonstrating reaction with immune serum, or with antisera produced against the protein itself.
  • detecting or “detected” as used herein mean using known techniques for detection of biologic molecules such as immunochemical or histological methods and refer to qualitatively or quantitatively determining the presence or concentration of the biomolecule under investigation.
  • isolated is meant a biological molecule free from at least some of the components with which it naturally occurs.
  • antibody or “antibodies” or “functional parts thereof as used herein is an art recognized term and is understood to refer to molecules or active fragments of molecules that bind to known antigens, particularly to immunoglobulin molecules and to immunologically active portions of immunoglobulin molecules, i.e molecules that contain a binding site that immunospecifically binds an antigen.
  • the immunoglobulin according to the invention can be of any type (IgG, IgM, IgD, IgE, IgA and IgY) or class (lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclasses of immunoglobulin molecule.
  • Antibodies are intended within the scope of the present invention to include monoclonal antibodies, polyclonal, chimeric, single chain, bispecific, simianized, human and humanized antibodies, camelid antibodies, diabodies, as well as functional parts or active fragments thereof.
  • active fragments of molecules that bind to known antigens include Fab and F(ab') 2 fragments, including the products of a Fab immunoglobulin expression library and epitope-binding fragments of any of the antibodies and fragments mentioned above.
  • These active fragments can be derived from an antibody of the present invention by a number of techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and subjected to HPLC gel filtration.
  • a “humanized antibody” refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s).
  • a humanized antibody may further refer to an antibody having a variable region where one or more of its framework regions have human or primate amino acids.
  • framework support residues may be altered to preserve binding affinity.
  • a “humanized antibody” may also be obtained by a novel genetic engineering approach that enables production of affinity-matured humanlike polyclonal antibodies in large animals such as, for example, rabbits (http://www.rctech.com/bioventures/therapeutic.php).
  • Fully human antibody or “human” antibody is meant to refer to an antibody derived from transgenic mice carrying human antibody genes or from human cells. To the human immune system, however, the difference between “fully human”, “human”, and “humanized” antibodies may be negligible or nonexistent and as such all three may be of equal efficacy and safety.
  • monoclonal antibody is also well recognized in the art and refers to an antibody that is mass produced in the laboratory from a single clone and that recognizes only one antigen.
  • Monoclonal antibodies are typically made by fusing a normally short-lived, antibody- producing B cell to a fast-growing cell, such as a cancer cell (sometimes referred to as an "immortal" cell). The resulting hybrid cell, or hybridoma, multiplies rapidly, creating a clone that produces large quantities of the antibody.
  • antigen refers to an entity or fragment thereof which can induce an immune response in an organism, particularly an animal, more particularly a mammal including a human.
  • the term includes immunogens and regions responsible for antigenicity or antigenic determinants.
  • the term “soluble” means partially or completely dissolved in an aqueous solution.
  • immunogenic refers to substances which elicit or enhance the production of antibodies, T-cells and other reactive immune cells directed against an immunogenic agent and contribute to an immune response in humans or animals.
  • An immune response occurs when an individual produces sufficient antibodies, T-cells and other reactive immune cells against administered immunogenic compositions of the present invention to moderate or alleviate the disorder to be treated.
  • hybrida is art recognized and is understood by those of ordinary skill in the art to refer to a cell produced by the fusion of an antibody-producing cell and an immortal cell, e.g. a multiple myeloma cell. This hybrid cell is capable of producing a continuous supply of antibody. See the definition of "monoclonal antibody” above and the Examples below for a more detailed description of the method of fusion.
  • carrier means a structure in which antigenic peptide or supramolecular construct can be incorporated into or can be associated with, thereby presenting or exposing antigenic peptides or part of the peptide to the immune system of a human or animal.
  • Any particle that can be suitably used in animal or human therapy such as, for example, a vesicle, a particle or a particulate body may be used as a carrier within the context of the present invention.
  • carrier further comprises methods of delivery wherein supramolecular antigenic construct compositions comprising the antigenic peptide may be transported to desired sites by delivery mechanisms.
  • delivery mechanisms utilizes colloidal metals such as colloidal gold.
  • Carrier proteins that can be used in the supramolecular antigenic construct compositions of the present invention include, but are not limited to, maltose binding peptide "MBP”; bovine serum albumin “BSA”; keyhole lympet hemocyanin “KLH”; ovalbumin; flagellin; thyroglobulin; serum albumin of any species; gamma globulin of any species; syngeneic cells; syngeneic cells bearing la antigens; and polymers of D- and/or L- amino acids.
  • terapéuticaally effective amount refers to the amount of binding peptide which, when administered to a human or animal, is sufficient to result in a therapeutic effect in said human or animal.
  • the effective amount is readily determined by one of ordinary skill in the art following routine procedures.
  • PHF neurofibrillary tangles of Alzheimer's Disease (AD) and neuropil threads. PHF may also be seen in some but not all dystrophic neurites associated with neuritic plaques.
  • PHF The major component of PHF is a hyperphosphorylated form of microtubule-associated protein tau.
  • PHF are composed of disulfide-linked antiparallel hyper-phosphorylated tau proteins.
  • PHF tau may be truncated of its C-terminal 20 amino acid residues. The mechanisms underlying PHF formation are uncertain but hyper- phosphorylation of tau may disengage it from microtubules, increasing the soluble pool of tau.
  • the antibody induced response to the antigenic composition according to the invention is largely T-cell independent.
  • a nude mouse model was used in this respect and nude mice were vaccinated and antibody responses measured to evaluate the ⁇ -specific antibody response induced by the antigenic composition according to the invention in the immunized nude mice.
  • the nude mice carry the Foxnl nu mutation and as a consequence, have reduced T-cell function due to the lack of a proper thymus.
  • a “pharmaceutically effective amount” as used herein refers to a dose of the active ingredient in a pharmaceutical composition adequate to cure, or at least partially arrest, the symptoms of the disease, disorder or condition to be treated or any complications associated therewith.
  • the present invention provides binding peptides recognizing and binding to major pathological phospho-epitopes of the tau protein.
  • the present invention provides specific antibodies against linear and conformational, simple and complex phospho-epitopes on protein tau that are believed to be responsible for synapto- and neuro-toxicity in tauopathies, including AD.
  • the present invention relates in one embodiment to a binding peptide or a functional part thereof, particularly to an antibody, particularly a monoclonal antibody or a functional part thereof, which binding peptide or antibody recognizes and specifically binds to a phospho-epitope on a mammalian, particularly on the human Tau protein or on a fragment thereof, particularly to a pathological protein tau conformer, but, in one embodiment, does not bind to the corresponding unphosphorylated epitope and/or to non-related epitopes, wherein said binding peptide or antibody has a high binding affinity with a dissociation constant of at least 10 nM, particularly of at least 8 nM, particularly of at least 5 nM, particularly of at least 2 nM, particularly of at least 1 nM, particularly of at least 500 pM, particularly of at least 400 pM particularly of at least 300 pM, particularly of at least 200 pM, particularly of at least 100 pM, particularly of at least 50 pM.
  • Soluble Tau protein refers to proteins consisting of both completely soiubiiized Tau protein/peptide monomers or of Tau-like peptides/proteins, or of modified or truncated Tau peptides/proteins or of other derivates of Tau peptides/proteins monomers, and of Tau protein oligomers.
  • Soluble Tau excludes particularly neurofibrillary tangles (NFT).
  • Insoluble Tau refers to multiple aggregated monomers of Tau peptides or proteins, or of Tau-like peptides/proteins, or of modified or truncated Tau peptides/proteins or of other derivates of Tau peptides/proteins forming oligomeric or polymeric structures which are insoluble both in vitro in aqueous medium and in vivo in the mammalian or human body more particularly in the brain, but particularly to multiple aggregated monomers of Tau or of modified or truncated Tau peptides/proteins or of derivatives thereof, which are insoluble in the mammalian or human body more particularly in the brain, respectively.
  • “Insoluble Tau” particularly includes neurofibrillary tangles (NFT).
  • “"Monomeric Tau” or “Tau monomer” as used herein refers to completely solubilized Tau proteins without aggregated complexes in aqueous medium.
  • Aggregated Tau refers to multiple aggregated monomers of Tau peptides or proteins , or of Tau-like peptides/proteins, or of modified or truncated Tau peptides/proteins or of other derivates of Tau peptides/proteins forming oligomeric or polymeric structures which are insoluble or soluble both in vitro in aqueous medium and in vivo in the mammalian or human body more particularly in the brain, but particularly to multiple aggregated monomers of Tau or of modified or truncated Tau peptides/proteins or of derivatives thereof, which are insoluble or soluble in the mammalian or human body more particularly in the brain, respectively.”
  • a “modulating antibody” refers to an antibody or a functional fragment thereof as described herein in the various embodiments, which may either up-regulate (e.g. , activate or stimulate), down-regulate (e.g., inhibit or suppress) or otherwise change a functional property, biological activity or level of soluble and/or insoluble and/or oligomeric Tau protein, particularly of soluble phosphorylated tau protein, in vivo, particularly in the brain, particularly in the brain cortex and/or hippocampus, of an animal, particularly a mammal or a human containing increased levels of soluble tau protein and/or soluble phosphorylated tau protein.
  • a modulating antibody or functional fragment thereof may act to modulate a tau protein or a polypeptide encoding said tau protein either directly or indirectly.
  • a modulating antibody or functional fragment thereof reduces the levels of soluble and/or insoluble and/or oligomeric, particularly soluble and insoluble tau protein, particularly soluble and insoluble and oligomeric tau protein.
  • the soluble and/or insoluble and/or oligomeric tau protein is phosphorylated tau protein, particularly soluble phosphorylated tau protein, in the brain, particularly in the brain cortex and/or hippocampus, of an animal, particularly a mammal or a human containing increased levels of tau protein and/or phosphorylated tau protein, particularly of soluble tau protein and/or soluble phosphorylated tau protein.”
  • the present invention provides a pharmaceutical composition comprising a binding peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide comprising a nucleic acid sequence encoding said binding peptide or antibody, according to any one of the embodiments described and claimed herein, or a combination thereof, in a therapeutically effective amount together with a pharmaceutically acceptable carrier.
  • Suitable pharmaceutical carriers, diluents and/or excipients are well known in the art and include, for example, phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, various types of wetting agents, sterile solutions, etc.
  • the binding peptides according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof, can be prepared in a physiologically acceptable formulation and may comprise a pharmaceutically acceptable carrier, diluent and/or excipient using known techniques.
  • a pharmaceutically acceptable carrier diluent and/or excipient
  • the binding peptides according to the invention and as described herein including any functionally equivalent binding peptides or functional parts thereof, in particular, the monoclonal antibodies of the invention including any functionally equivalent antibodies or functional parts thereof are combined with a pharmaceutically acceptable carrier, diluent and/or excipient to form a therapeutic composition.
  • Suitable pharmaceutical carriers, diluents and/or excipients are well known in the art and include, for example, phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, various types of wetting agents, sterile solutions, etc.
  • Formulation of the pharmaceutical composition according to the invention can be accomplished according to standard methodology know to those of ordinary skill in the art.
  • compositions of the present invention may be administered to a subject in the form of a solid, liquid or aerosol at a suitable, pharmaceutically effective dose.
  • solid compositions include pills, creams, and implantable dosage units. Pills may be administered orally.
  • Therapeutic creams may be administered topically.
  • Implantable dosage units may be administered locally, for example, at a tumor site, or may be implanted for systematic release of the therapeutic composition, for example, subcutaneously.
  • liquid compositions include formulations adapted for injection intramuscularly, subcutaneously, intravenously, intra-arterially, and formulations for topical and intraocular administration.
  • aerosol formulations include inhaler formulations for administration to the lungs.
  • compositions may be administered by standard routes of administration.
  • the composition may be administered by topical, oral, rectal, nasal, interdermal, intraperitoneal, or parenteral (for example, intravenous, subcutaneous, or intramuscular) routes.
  • composition may be incorporated into sustained release matrices such as biodegradable polymers, the polymers being implanted in the vicinity of where delivery is desired, for example, at the site of a tumor.
  • sustained release matrices such as biodegradable polymers
  • the method includes administration of a single dose, administration of repeated doses at predetermined time intervals, and sustained administration for a predetermined period of time.
  • a sustained release matrix is a matrix made of materials, usually polymers which are degradable by enzymatic or acid/base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids.
  • the sustained release matrix desirably is chosen by biocompatible materials such as liposomes, polylactides (polylactide acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.
  • the dosage of the composition will depend on various factors such as, for example, the condition of being treated, the particular composition used, and other clinical factors such as weight, size, sex and general health condition of the patient, body surface area, the particular compound or composition to be administered, other drugs being administered concurrently, and the route of administration.
  • composition according to the invention may be administered in combination with other compositions comprising an biologically active substance or compound such as, for example, a known compound used in the medication of tauopathies and/or of amyloidoses, a group of diseases and disorders associated with amyloid or amyloid-like protein such as the amyloid ⁇ protein involved in Alzheimer's Disease.
  • an biologically active substance or compound such as, for example, a known compound used in the medication of tauopathies and/or of amyloidoses, a group of diseases and disorders associated with amyloid or amyloid-like protein such as the amyloid ⁇ protein involved in Alzheimer's Disease.
  • the other biologically active substance or compound may exert its biological effect by the same or a similar mechanism as the therapeutic vaccine according to the invention or by an unrelated mechanism of action or by a multiplicity of related and/or unrelated mechanisms of action.
  • the other biologically active compound may include neutron-transmission enhancers, psychotherapeutic drugs, acetylcholine esterase inhibitors, calcium-channel blockers, biogenic amines, benzodiazepine tranquillizers, acetylcholine synthesis, storage or release enhancers, acetylcholine postsynaptic receptor agonists, monoamine oxidase-A or - B inhibitors, N-methyl-D-aspartate glutamate receptor antagonists, non-steroidal antiinflammatory drugs, antioxidants, and serotonergic receptor antagonists.
  • neutron-transmission enhancers may include neutron-transmission enhancers, psychotherapeutic drugs, acetylcholine esterase inhibitors, calcium-channel blockers, biogenic amines, benzodiazepine tranquillizers, acetylcholine synthesis, storage or release enhancers, acetylcholine postsynaptic receptor agonists, monoamine oxidase-A or - B inhibitors, N
  • the biologically active agent or compound may comprise at least one compound selected from the group consisting of compounds against oxidative stress, anti-apoptotic compounds, metal chelators, inhibitors of DNA repair such as pirenzepin and metabolites, 3- amino-1 -propanesulfonic acid (3APS), 1 ,3-propanedisulfonate (1.3PDS), secretase activators, [beta]- and 7-secretase inhibitors, tau proteins, neurotransmitter, /3-sheet breakers, antiinflammatory molecules, "atypical antipsychotics” such as, for example clozapine, ziprasidone, risperidone, aripiprazole or olanzapine or cholinesterase inhibitors (ChEls) such as tacrine, rivastigmine, donepezil, and/or galantamine and other drugs and nutritive supplements such as, for example, vitamin B 12, cysteine, a precursor of acetylcholine, lecithin,
  • composition according to the invention may comprise niacin or memantine together with a binding peptide according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof, and, optionally, a pharmaceutically acceptable carrier and/or a diluent and/or an excipient.
  • compositions that comprise "atypical antipsychotics" such as, for example clozapine, ziprasidone, risperidone, aripiprazole or olanzapine for the treatment of positive and negative psychotic symptoms including hallucinations, delusions, thought disorders (manifested by marked incoherence, derailment, tangentiality), and playful or disorganized behavior, as well as anhedonia, flattened affect, apathy, and social withdrawal, together with the binding peptide according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof, and, optionally, a pharmaceutically acceptable carrier and/or a diluent and/or an excipient.
  • "atypical antipsychotics” such as, for example clozapine, ziprasidone, risperidone, aripiprazole or olanzapine for the treatment of positive and negative psychotic symptoms including hallucinations, delusions, thought disorders (manifested by marked incoher
  • compositions in addition to the binding peptide according to the invention are those disclosed, for example, in WO 2004/058258 (see especially pages 16 and 17) including therapeutic drug targets (page 36-39), alkanesulfonic acids and alkanolsulfuric acid (pages 39-51 ), cholinesterase inhibitors (pages 51-56), NMDA receptor antagonists (pages 56-58), estrogens (pages 58-59), non-steroidal antiinflammatory drugs (pages 60-61 ), antioxidants (pages 61 -62), peroxisome proliferators- activated receptors (PPAR) agonists (pages 63-67), cholesterol-lowering agents (pages 68- 75); amyloid inhibitors (pages 75-77), amyloid formation inhibitors (pages 77-78), metal chelators (pages 78-79), anti-psychotics and anti-depressants (pages 80-82), nutritional supplements (pages 83-89) and compounds increasing the
  • Proteinaceous pharmaceutically active matter may be present in amounts between 1 ng and 10 mg per dose.
  • the regime of administration should be in the range of between 0.1 pg and 10 mg of the antibody according to the invention, particularly in a range 1.0 pg to 1.0 mg, and more particularly in a range of between 1.0 Mg and 100 M9 ⁇ with all individual numbers falling within these ranges also being part of the invention. If the administration occurs through continuous infusion a more proper dosage may be in the range of between 0.01 Mg and 10 mg units per kilogram of body weight per hour with all individual numbers falling within these ranges also being part of the invention.
  • Parenteral administration will generally be parenterally, e.g. intravenously or subcutaneously.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions.
  • Non-aqueous solvents include, without being limited to, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous solvents may be chosen from the group consisting of water, alcohol/aqueous solutions, emulsions or suspensions including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose) and others. Preservatives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, etc.
  • the pharmaceutical composition may further comprise proteinaceous carriers such as, for example, serum albumin or immunoglobulin, particularly of human origin. Further biologically active agents may be present in the pharmaceutical composition of the invention dependent on its the intended use.
  • proteinaceous carriers such as, for example, serum albumin or immunoglobulin, particularly of human origin.
  • Further biologically active agents may be present in the pharmaceutical composition of the invention dependent on its the intended use.
  • certain embodiments of the invention provide for the binding peptide according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof, to traverse the blood-brain barrier.
  • Certain neurodegenerative diseases are associated with an increase in permeability of the blood-brain barrier, such that the binding peptide according to the invention including antibodies, particularly monoclonal antibodies or active fragment thereof can be readily introduced to the brain.
  • the blood-brain barrier remains intact, several art-known approaches exist for transporting molecules across it, including, but not limited to, physical methods, lipid-based methods, and receptor and channel-based methods.
  • Circumvention methods include, but are not limited to, direct injection into the brain (see, e.g., Papanastassiou et al. , Gene Therapy 9: 398-406 (2002)) and implanting a delivery device in the brain (see, e.g., Gill et al., Nature Med. 9: 589-595 (2003); and Gliadel Wafers (TM), Guildford Pharmaceutical).
  • Methods of creating openings in the barrier include, but are not limited to, ultrasound (see, e.g., U.S. Patent Publication No. 2002/0038086), osmotic pressure (e.g., by administration of hypertonic mannitol (Neuwelt, E. A., Implication of the Blood-Brain Barrier and its Manipulation, Vols 1 & 2, Plenum Press, N. Y. (1989)), permeabilization by, e.g., bradykinin or permeabilizer A-7 (see, e.g., U.S. Patent Nos.
  • Lipid-based methods of transporting the binding peptide according to the invention including antibodies, particularly monoclonal antibodies, or an active fragment thereof across the blood-brain barrier include, but are not limited to, encapsulating the binding peptide according to the invention including antibodies, particularly monoclonal antibodies, or active fragment thereof in liposomes that are coupled to active fragments thereof that bind to receptors on the vascular endothelium of the blood-brain barrier (see, e.g. , U.S. Patent Application Publication No. 20020025313), and coating the binding peptide according to the invention including antibodies, particularly monoclonal antibodies, or active fragment thereof in low-density lipoprotein particles (see, e.g., U.S. Patent Application Publication No. 20040204354) or apolipoprotein E (see, e.g. , U.S. Patent Application Publication No. 20040 31692).
  • Receptor and channel-based methods of transporting the binding peptide according to the invention including antibodies, particularly monoclonal antibodies, or active fragment thereof across the blood-brain barrier include, but are not limited to, using glucocorticoid blockers to increase permeability of the blood-brain barrier (see, e.g., U.S. Patent Application Publication Nos. 2002/0065259, 2003/0162695, and 2005/0124533); activating potassium channels (see, e.g., U.S. Patent Application Publication No. 2005/0089473), inhibiting ABC drug transporters (see, e.g., U.S. Patent Application Publication No.
  • Single or repeated administrations of the binding peptide according to the invention including antibodies, particularly monoclonal antibodies, or an active fragment thereof, or of a pharmaceutical composition according to the invention may be provided to a subject over an extended period of time.
  • the duration of administration may be between 1 week and up to 12 month or more.
  • the binding peptide, antibody or pharmaceutical composition may be administerd once a week, once every two weeks, three weeks, four weeks, etc, or at a higher or lower frequency depending on the needs of the subject to be treated.
  • the present invention provides methods and kits for the detection and diagnosis of tau-protein-associated diseases, disorders or conditions, including neurodegenerative diseases or disorders such as tauopathies comprising a heterogeneous group of neurodegenerative diseases or disorders including diseases or disorders which manifest both tau and amyloid pathologies including, but not limited to, Alzheimer's Disease, Creutzfeldt-Jacob disease, Dementia pugilistica, Down's Syndrome, Gerstmann-Straussler- Scheinker disease, inclusion-body myositis, and prion protein cerebral amyloid angiopathy, traumatic brain injury and further of diseases or disorders which do not show a distinct amyloid pathology including, but not limited to, amyotrophic lateral sclerosis/parkinsonism- dementia complex of Guam, Non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, front
  • the present invention provides methods and kits for diagnosing a predisposition to tau-protein-associated diseases, disorders or conditions, including neurodegenerative diseases or disorders such as tauopathies comprising a heterogeneous group of neurodegenerative diseases or disorders including diseases or disorders which show co- manifest both tau and amyloid pathologies, or for monitoring minimal residual disease in a patient or for predicting responsiveness of a patient to a treatment with a binding peptide according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof, or a composition according to the invention and as described herein.
  • These methods include known immunological methods commonly used for detecting or quantifying substances in biological samples or in an in situ condition.
  • Diagnosis of a tau-protein-associated disease or condition or of a predisposition to an tau- protein-associated disease or condition in a subject in need thereof, particularly a mammal, more particularly a human, including neurodegenerative diseases or disorders such as tauopathies comprising a heterogeneous group of neurodegenerative diseases or disorders including diseases or disorders which manifest both of tau and amyloid pathologies, may be achieved by detecting the immunospecific binding of a binding peptide of the invention, particularly of an antibody, particularly of a monoclonal antibody or an active fragment thereof, to an epitope of the tau protein in a sample or in situ, which includes bringing the sample or a specific body part or body area suspected to contain the tau protein into contact with an antibody which binds an epitope of the tau protein, allowing the antibody to bind to the tau protein to form an immunologic complex, detecting the formation of the immunologic complex and correlating the presence or absence of the immunologic complex with the presence or absence of tau protein in the sample or specific body part or area, optional
  • Monitoring minimal residual disease in a subject, particularly a mammal, more particularly a human, following treatment with a binding peptide according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof, or a composition according to the invention may be achieved by detecting the immunospecific binding of a binding peptide of the invention, particularly of an antibody, particularly a monoclonal antibody or an active fragment thereof to an epitope of the tau protein in a sample or in situ, which includes bringing the sample or a specific body part or body area suspected to contain the tau protein into contact with a binding peptide according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof, which binds an epitope of the tau protein, allowing the binding peptide according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof, to bind to the tau protein to form an immunologic complex, detecting the formation of the immunologic complex and correlating the presence or absence of the immunologic complex with the presence or absence of tau protein in the sample or specific body part or area
  • Predicting responsiveness of a subject, particularly a mammal, more particularly a human, to a treatment with a binding peptide according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof, or a composition according to the invention may be achieved by detecting the immunospecific binding of a binding peptide, particularly of a monoclonal antibody or an active fragment thereof to an epitope of the tau protein in a sample or in situ, which includes bringing the sample or a specific body part or body area suspected to contain the tau protein into contact with a binding peptide according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof, which binds an epitope of the tau protein, allowing the binding peptide according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof, to bind to the tau protein to form an immunologic complex, detecting the formation of the immunologic complex and correlating the presence or absence of the immunologic complex with the presence or absence of tau protein in the sample or specific body part or area, optionally
  • fluids such as serum, plasma, saliva, gastric secretions, mucus, cerebrospinal fluid, lymphatic fluid and the like or tissue or cell samples obtained from an organism such as neural, brain,
  • any immunoassay known to those of ordinary skill in the art may be used such as, for example, assays which utilize indirect detection methods using secondary reagents for detection, ELISA's and immunoprecipitation and agglutination assays.
  • assays which utilize indirect detection methods using secondary reagents for detection, ELISA's and immunoprecipitation and agglutination assays.
  • a detailed description of these assays is, for example, given in Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New York 1988 555-612, WO96/13590 to Maertens and Stuyver, Zrein et al, (1998) and WO96/29605.
  • the binding peptide according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof, of the invention or any active and functional part thereof may be administered to the organism to be diagnosed by methods known in the art such as, for example, intravenous, subcutaneous, intranasal, intraperitoneal, intracerebral, intraarterial injection such that a specific binding between an antibody according to the invention with an eptitopic region on the amyloid protein may occur.
  • the binding peptide/antigen complex may conveniently be detected through a label attached to the binding peptide according to the invention including antibodies, particularly monoclonal antibodies, or a functional fragment thereof or any other art- known method of detection.
  • the immunoassays used in diagnostic applications or in applications for diagnosing a predisposition to a tau protein-associated disease or condition including neurodegenerative diseases or disorders such as tauopathies comprising a heterogeneous group of neurodegenerative diseases or disorders including diseases or disorders which manifest both tau and amyloid pathologies, or for monitoring minimal residual disease in a patient or for predicting responsiveness of a patient to a treatment with a binding peptide according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof, or a composition according to the invention and as described herein typically rely on labelled antigens, binding peptides, or secondary reagents for detection.
  • proteins or reagents can be labelled with compounds generally known to those of ordinary skill in the art including enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances including, but not limited to colored particles, such as colloidal gold and latex beads.
  • enzymes radioisotopes
  • fluorescent, luminescent and chromogenic substances including, but not limited to colored particles, such as colloidal gold and latex beads.
  • radioactive labelling can be used for almost all types of assays and with most variations.
  • Enzyme-conjugated labels are particularly useful when radioactivity must be avoided or when quick results are needed.
  • Fluorochromes although requiring expensive equipment for their use, provide a very sensitive method of detection. Binding peptides useful in these assays are those disclosed claimed herein including antibodies, particularly monoclonal antibodies, polyclonal antibodies, and affinity purified polyclonal antibodies.
  • the binding peptide according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof may be labelled indirectly by reaction with labelled substances that have an affinity for immunoglobulin, such as protein A or G or second antibodies.
  • the binding peptide according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof may be conjugated with a second substance and detected with a labelled third substance having an affinity for the second substance conjugated to the antibody.
  • the binding peptide according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof may be conjugated to biotin and the binding peptide/biotin conjugate detected using labelled avidin or streptavidin.
  • the binding peptide may be conjugated to a hapten and the binding peptide/hapten conjugate detected using labelled anti-hapten binding peptide.
  • the antibody is labeled indirectly by reactivity with a second antibody that has been labeled with a detectable label.
  • the second antibody is preferably one that binds to antibodies of the animal from which the monoclonal antibody is derived. In other words, if the monoclonal antibody is a mouse antibody, then the labeled, second antibody is an anti- mouse antibody.
  • this label is preferably an antibody-coated bead, particularly a magnetic bead.
  • the label is preferably a detectable molecule such as a radioactive, fluorescent or an electrochemiluminescent substance.
  • an alternative double antibody system often referred to as fast format systems because they are adapted to rapid determinations of the presence of an analyte, may also be employed within the scope of the present invention.
  • the system requires high affinity between the antibody and the analyte.
  • the presence of the amyloid protein is determined using a pair of antibodies, each specific for amyloid protein.
  • One of said pairs of antibodies is referred to herein as a "detector antibody” and the other of said pair of antibodies is referred to herein as a "capture antibody”.
  • the monoclonal antibody of the present invention can be used as either a capture antibody or a detector antibody.
  • the monoclonal antibody of the present invention can also be used as both capture and detector antibody, together in a single assay.
  • One embodiment of the present invention thus uses the double antibody sandwich method for detecting amyloid protein in a sample of biological fluid.
  • the analyte (amyloid protein) is sandwiched between the detector antibody and the capture antibody, the capture antibody being irreversibly immobilized onto a solid support.
  • the detector antibody would contain a detectable label, in order to identify the presence of the antibody-analyte sandwich and thus the presence of the analyte.
  • Exemplary solid phase substances include, but are not limited to, microtiter plates, test tubes of polystyrene, magnetic, plastic or glass beads and slides which are well known in the field of radioimmunoassay and enzyme immunoassay. Methods for coupling antibodies to solid phases are also well known to those of ordinary skill in the art. More recently, a number of porous material such as nylon, nitrocellulose, cellulose acetate, glass fibers and other porous polymers have been employed as solid supports.
  • the present invention also relates to a diagnostic kit for detecting tau protein in a biological sample comprising a composition as defined above. Moreover, the present invention relates to the latter diagnostic kit which, in addition to a composition as defined above, also comprises a detection reagent as defined above.
  • diagnostic kit refers in general to any diagnostic kit known in the art. More specifically, the latter term refers to a diagnostic kit as described in Zrein et al. (1998).
  • the binding peptides are directly or indirectly attached to a suitable reporter molecule, e.g., an enzyme or a radionuclide.
  • the test kit includes a container holding one or more binding peptides according to the present invention and instructions for using the binding peptides for the purpose of binding to tau antigen to form an immunologic complex and detecting the formation of the immunologic complex such that presence or absence of the immunologic complex correlates with presence or absence of tau protein.
  • Hybridomas were generated by fusion of tau vaccine immunized mouse spleen cells with a myeloma cell line. The hybridomas were assessed for reactivity against both phosphorylated and non-phosphorylated full-length Tau protein, as well as the phosphorylated and non-phosphorylated Tau antigenic peptides used in the vaccine preparation. Hybridoma screening was also performed for reactivity of hybridomas supernatant for tau tangles using immunochemistry on Tau transgenic mouse brain slices.
  • a wild type C57BL/6 mouse vaccinated with ACI-35 (Tau393-408 [pS396, pS404]) was used for hybridoma production.
  • the mouse was boosted with ACI-35 vaccine on day 0 then again on day 4 and the fusion was performed on day 7.
  • splenocytes from the immunized mouse were fused with 2 x 10 7 SP2-0- Ag14 myeloma cells at a ratio of 3 splenocytes / 1 myeloma cell.
  • the fusions resulted in 8x96 well plates and the clones were named according to the plate (1-8) then the row (A-G) and finally the column (1-12).
  • ELISA plates (Costar; Sigma) were coated with 50 ⁇ /well of anti-mouse IgG antibody (AbD Serotec, Dusseldorf, Germany) in coating buffer for 16 hr at 4°C. After washing plates with PBS/Tween, wells were blocked with 100 ⁇ /well of blocking solution for 1 hr at ambient temperature. Undiluted hybridoma supernatants (50 ⁇ per well) were incubated for 1 hr at ambient temperature.
  • HRP horseradish peroxidase
  • Hybridomas ELISA screen was performed on pTau peptide (ACI-35, T3.5: Tau393- 408[pS396/pS404; Polypeptide Laboratories, Hillerod, Denmark), the corresponding non- phosphory!ated Tau peptide (T3.6: Tau393-408, Polypeptide Laboratories), phosphorylated full-length (441 aa) Tau protein (pTau protein, Vandebroek et al., 2005) and full-length (441 aa) Tau protein (Tau protein, SignalChem, Richmond, Canada). Finally Bovine Serum Albumin (BSA) was used as negative control.
  • BSA Bovine Serum Albumin
  • Plates were coated with 10 pg/ml of corresponding Tau peptide and 1 g/ml of corresponding Tau protein overnight at 4°C. After washing each well with PBS-0.05% Tween 20 and blocking with 1 % BSA in PBS-0.05% Tween 20, undiluted hybridoma supernatant or medium negative control were added to the plates and incubated at 37°C for 2 hours. After washing plates were incubated with an alkaline phosphatase (AP)-conjugated anti-mouse IgG total antibody (Jackson Laboratories, Baltimore, PA, USA) for 2 hours at 37°C.
  • AP alkaline phosphatase
  • Hybridomas IHC screen Binding of anti-Tau antibodies to tangles in brain sections from transgenic mice (TAUPIR) TAUPIR experiments were done according to protocol from EXAMPLE 3.1.2.
  • ELISA plates were coated with 5ug/ml of anti-mouse IgG F(ab')2 fragment specific antibody (Jackson Laboratories, Baltimore, PA, USA) in carbonate-bicarbonate coating buffer pH 9.6 (Sigma, Buchs, Switzerland) overnight at 4°C. After washing plates, undiluted hybridoma supernatant, positive control lgG1 antibody (6E10 at 1 ug/ml: Covance, Emeryville, CA, USA) or negative control (culture medium alone) were incubated for 1 hr at RT.
  • anti-mouse IgG F(ab')2 fragment specific antibody Jackson Laboratories, Baltimore, PA, USA
  • carbonate-bicarbonate coating buffer pH 9.6 Sigma, Buchs, Switzerland
  • the secondary AP-conjugated goat anti-mouse IgG (subclasses 1 +2a+2b+3) Fey fragment specific antibody (Jackson Laboratories, Baltimore, PA, USA) was incubated on the plates for 2 hrs at 37°C. After a final washing, detection was performed with pNPP (para- nitro-phenyl-phosphate), the phosphatase substrate for AP, and plates were read at 405 nm using an ELISA plate reader. Results are expressed as O.D. (Optical Density).
  • the cell supernatants from the 8x96 well plates resulting from the fusion were screened for production of IgG.
  • Out of 768 wells (8x96 wells) tested, 48 wells positive for IgG production were selected based on the best binding to the vaccine phospho-peptide, and to full-length phospho-Tau. Selection was based on binding to the peptide and full-length phospho-Tau protein by ELISA, and also to selectivity when comparing to non-phospho-peptide and non- phospho full-length Tau protein.
  • 24 selected hybridomas were subcloned by seeding 2 plates per hybridoma at 1 cell/well and 1 plate at 0.5 cell/well.
  • the antibodies generated have shown high specificity to pTau peptides with only marginal binding to non-phosphorylated peptides.
  • Antibody heavy and light variable region genes from the hybridoma cells are cloned and the DNA sequences and location of the complementarity determining regions (CDRs) determined as well as the antibodies binding features.
  • CDRs complementarity determining regions
  • V H and VK cDNAs were prepared using reverse transcriptase with IgG and kappa constant region primers.
  • the first strand cDNAs were amplified by PGR using a large set of signal sequence primers.
  • the amplified DNAs were gel-purified and cloned into the vector pGem ® T Easy (Promega).
  • the V H and V K clones obtained were screened for inserts of the expected size.
  • the DNA sequence of selected clones was determined in both directions by automated DNA sequencing. The locations of the complementarity determining regions (CDRs) in the sequences were determined with reference to other antibody sequences (Kabat EA et a/., 1991 ).
  • the objective was to measure the phospho-Tau (pTau) binding of the antibodies generated from subcloned hybridomas derived from mice immunized with the tau liposomal vaccines.
  • an enzyme-linked immunosorbant assay (ELISA) was used to measure the binding of the purified antibodies to both phosphorylated and non-phosphorylated full-length Tau protein, as well as the phosphorylated and non-phosphorylated Tau antigenic peptides used for the liposomal vaccine preparation.
  • the screening was completed by two other methods. Immunohistochemistry (IHC) on brain sections from a Tau transgenic animal (TAUPIR) using an anti-tau antibody as the primary antibody was done. Additionally, a western blot (WB) on brain protein homogenates from Tau transgenic mice was performed, using an anti-tau antibody as the blotting antibody.
  • IHC Immunohistochemistry
  • WB western blot
  • an ELISA assay was used. Briefly, Nunc MaxiSorp 96-well plates (Nunc, Rosktlde, Denmark) were coated with 1 pg/mL of full-length (441 aa) Tau protein (SignalChem, Richmond, Canada) or phosphorylated full- length (441 aa) Tau protein (Vandebroek et al., 2005). Additionally, plates were coated with 10 g/mL of the Tau-derived vaccine peptide, Tau393-408 (phosphorylated or not on S396 and S404).
  • the antibody being tested was then added in an 8 or 16 twofold dilution series between 20 and 0 pg/mL, and allowed to incubate for 2 hr at 37°C. Plates were then washed as described previously, and AP-conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, Suffolk, England) was added at 1/6000 dilution in 0.05% Tween20/PBS for 2 hr at 37°C.
  • pNPP p- nitrophenyl phosphate disodium hexahydrate
  • TAUPIR and Western-blots Binding of anti-Tau antibody to Tau tangles in brain sections from a Tau transgenic animal (TAUPIR)
  • mice transgenic mice expressing the longest isoform (441 aa) of hTau P301L
  • old (>18 months old) double transgenic biGT GSK-3p transgenic mice crossed with TPLH
  • double transgenic biAT hAPP V7171 transgenic mice crossed with TPLH mice
  • Tau knock-out mice TKO; 6 months old
  • Brain sections were washed for 5 min in PBS then incubated for 15 min at RT in 1.5% H 2 0 2 in PBS:MeOH (1 : 1 ) to block endogenous peroxidase activity.
  • Sections were next washed 3 times in PBST before incubation with an HRP-conjugated goat anti-mouse (purchased from Dako, Glostrup, Denmark) secondary antibody in PBST/10% FCS for 1 hour at RT.
  • HRP-conjugated goat anti-mouse purchased from Dako, Glostrup, Denmark
  • PBST/10% FCS PBST/10% FCS
  • Detection was done by incubating the sections for 3 min in Diaminobenzidine (DAB: 1 tablet in 10 ml of 50 mM Tris.HCI + 3 pi H 2 0 2 30%; MP Biomedicals. Solon, OH, USA).
  • DAB Diaminobenzidine
  • Sections were then transferred onto silanized glass- plates and air-dried on warm-plate at 50°C for 2 hours. Counterstaining was done using incubation with Mayers hematoxylin (Fluka Chemie, Buchs, Switzerland) for 1 min, followed by a washing step for 4 min in running tap-water. Sections were dehydrated by passing in 50%, 70%, 90% and twice in 100% ethanol bath then in Xylol 2 times for 1 min. Finally sections were mounted with DePeX (BDH Chemicals Ltd., Poole, England) under glass cover-slips for imaging.
  • Antibody ACI-35-2A1-Ab1 , ACI-35-2A1-Ab2, ACI-35-1 D2-Ab1 , ACI-35-2G5-Ab2 and ACI-35- 2G5-Ab3 demonstrated high binding activity and specificity to phosphorylated human Tau protein (Table 2), more specifically to the antigenic phospho-Tau peptide used in the corresponding vaccine. No cross-reactivity to non-phosphorylated Tau was observed, or to other phosphorylated and non-phosphorylated Tau-derived peptides tested.
  • Antibody ACI- 35-4A6-Ab1 displayed high binding activity only to the antigenic phospho-Tau peptide used in the vaccine preparation.
  • TAUPIR and WBs were used to look at binding to Tau tangles in brains of mice with advanced tauopathy (biGT > 18 months), and to full-length Tau in denatured homogenates derived from these mice.
  • Different brain regions were analyzed: cortex and CA1 , CA3 and dentate gyrus (DG) part of the hippocampus.
  • Antibodies ACI-35-2A1-Ab1 and ACI-35-2A1 - Ab2 displayed the best TAUPIR results with a dense cytoplasmic staining and clear neuropil threads, especially in the CA1 and CA3 regions of the hippocampus.
  • Antibody ACI-35-4A6- Ab1 was negative in TAUPIR with only faint sporadic tangle like structures lightly stained.
  • Antibody ACI-35-1 D2-Ab1 showed a good cytoplasmic TAUPIR staining with neuropil threads in the CA1 region.
  • Antibody ACI-35-2G5-Ab1 was negative in TAUPIR with nuclear staining and only some tangle staining.
  • ACI-35-2G5-Ab2 and ACI-35-2G5-Ab3 antibodies displayed similar good cytoplasmic TAUPIR staining with neuropil threads observed in the CA1 and CA3 of the hippocampus. The rating of staining quality using + or - signs is shown in Table 2.
  • the binding of the anti-tau antibodies to the phosphorylated Tau peptide was monitored in real-time using SPR. Analyses of the association and dissociation phases of antibody binding could be used to determine the association rate constant (k a ), dissociation rate constant (/ d ) as well as dissociation constant K D .
  • EXAMPLE 5 Binding Studies 111 ELISA on human brain samples (ELISA for detection of multimers of phosphorylated Tau)
  • Temporal post-mortem cortex for ten AD and ten age-matched controls were homogenized according to the following protocol. Brain fragments were weighted and homogenized in 9 volumes of 25 mM Tris-HCI pH 7.4, 150 mM NaCI, 1 mM EDTA, 1 mM EGTA containing phosphatase inhibitors (30 mM NaF, 0.2 mM Na3V04, 1 nM okadaic acid, 1 mM PMSF, 5 mM Na4P207) and protease inhibitors (Complete Mini; Roche, Switzerland). Homogenization was done on ice using a glass potter. This constitutes the Total Homogenate fraction (TH). Protein concentrations were measured using Bradford reagent (Sigma).
  • Setup 1 ELISA Setup 1 ELISA assay to detect the presence of multimers of phosphorylated Tau in human post-mortem cortical brain homogenates from AD-affected individuals and age-matched controls
  • Multititer 96-well plates were coated with antibodies overnight at 4 °C at 5 g/ml in carbonate/bicarbonate buffer. After 4 washes in PBS-Tween, plates were saturated with PBS-Tween 10% BSA for 1 hr at 37°C. Brain homogenates were then added to the wells at a concentration of 100 ng/ ⁇ . in 50 ⁇ _ PBS, and incubated for 2 hr at 37°C.
  • Antibodies ACI-35-2A1-Ab1 and ACI-35-2G5-Ab3 were tested for their ability to detect phosphoTau (pTau) multimers in brain homogenates from AD and control subjects, using a phospho- and multimer-specific Setup 1 ELISA.
  • pTau phosphoTau
  • We observed a highly significant (p ⁇ 0.001 ) difference between AD and age-matched controls (n 10) in this assay for both antibodies ( Figure 1 ).
  • Using human post-mortem cortical homogenates from AD and age-matched control brain we demonstrated the ability of ACI-35-2A1 -Ab1 and ACI-35-2G5-Ab3 anti-pTau antibodies to detect multimers of Tau-pS396 in post-mortem human brain samples.
  • EXAMPLE 6 Binding Studies IV - Western-blots on human brain samples. 6.1 Methods
  • Western-blots Western-blot assay to detect the presence of phosphorylated Tau in human post-mortem cortical brain homogenates from AD-affected individuals and age- matched controls
  • the anti-human Tau antibodies used in this study were the mouse ACI-35-2A1-Ab1 , ACI-35- 1 D2-Ab1 , and ACI-35-2G5-Ab3, all directed against Tau-pS396.
  • the mouse monoclonal TAU-13 antibody (Abeam ab24636) directed against total human Tau
  • the rabbit monoclonal antibody E178 directed against Tau-pS396 (Abeam ab32057) were used as controls.
  • 20 Mg of each total homogenate was loaded per lane of on a 10% polyacrylamide Bis-TRIS precast gel (Nupage Novex 10% Bis-TRIS Midi Gel, Invitrogen), Proteins were resolved as recommended by the manufacturer in NuPAGE MOPS SDS running buffer (Invitrogen NP0001 ).
  • Protein blotting was done for 3 hr in 25 mM TRIS pH 8.6, 190 mM Glycin buffer, 20% methanol, on ice on PVDF membranes (Immobilon-FL, Millipore IPFL00010). Membranes were blocked for 1 hr in Licor blocking buffer (Odyssey) diluted 1/3 in PBS.
  • Membranes were incubated overnight with primary antibodies at the following concentrations: TAU-13 at 0.6 pg/mL, E178 diluted 1/5000, ACI-35-2A1-Ab1 at 0.53 pg/mL, ACI-35-1 D2-Ab1 at 0.47 pg/mL, and ACI-35-2G5-Ab3 at 0.5 pg/mL, diluted 1/3 in Licor buffer and 2/3 PBS with 0.1 % Tween-20 (PBS-T).
  • membranes were incubated with a goat anti-mouse antibody coupled with the LICOR 800 dye (Goat anti- mouse IRDye 800 CW, Odyssay) for 1 hr at room temperature, washed again 4 times with PBS-T, and scanned for image reproduction using the LICOR system.
  • a goat anti-mouse antibody coupled with the LICOR 800 dye (Goat anti- mouse IRDye 800 CW, Odyssay) for 1 hr at room temperature
  • ACI-35- 2A1 -Ab1 , ACI-35-1 D2-AM , and ACI-35-2G5-Ab3 can discriminate between AD and age- matched controls for the presence of pS396 Tau in human post-mortem cortex, and thus these monoclonal antibodies recognize AD-associated pathological Tau isoforms.
  • soluble Tau (S1 ) fraction used for the AlphaLISA assay half volume of TH fraction was aliquoted and stored at -80°C. The remainder of the TH fraction was further processed by adding Triton X-100 to a final concentration of 0.4%. Samples were mixed well and vortexed several times before being centrifuged at 5 ⁇ 00 rpm for 5 min at 4°C. The supernatant constitutes the S1 fraction. The samples were aliquoted and stored at -80°C. Protein concentrations were measured using Bradford reagent.
  • AlphaLISA AlphaLISA assay to detect the presence phosphorylated Tau in human post-mortem cortical brain homogenates from AD-affected individuals and age-matched controls.
  • the biotinylated antibodies are designated as ACI-35-2A1-Ab1 - BT, ACI-35-1 D2-Ab1-BT, and ACI-35-2G5-Ab3-BT.
  • Antibody Tau-13 was conjugated to the activated Alpha Acceptor beads (Perkin Elmer) using the following protocol: 0.1 mg of Tau- 13 antibody solution (purified on Protein A column) was mixed with 1 mg of AlphaLISA Acceptor Bead pellets and complemented with 0.13 M phosphate buffer (pH 8.0) to a final reaction volume of 200 pL.
  • AlphaLISA is a homogenous assay based on bead proximity chemiluminescence. If Alpha Donor and Acceptor beads are in close proximity, upon laser excitation, a cascade of chemical reactions produces an amplified signal. Upon excitation at 680 nm, the photosensitizer contained in Donor beads converts ambient oxygen into more reactive singlet oxygen species. These singlets diffuse (up to 200 nm, within 4 psec of a half-life) and produce a chemiluminescent reaction in the Acceptor beads, leading to light emission.
  • the assay setting was as follows:
  • S1 samples were pre-diiuted in Alpha Assay buffer (PerkinElmer AL000C) to obtain a 20 pg/mL stock concentration.
  • the following reagents were added to a 384-well white OptiPlate (PerkinElmer) to a final volume of 50 pL: S1 brain homogenate (5 ⁇ _), 10 pL of ACI-35-2A1- Ab1 -BT, ACI-35-1 D2-Ab1-BT, or ACI-35-2G5-Ab3-BT for a final antibody concentration of 0.2 nM, 0.5 nM, or 0.5 nM, respectively, and 10 pL of Tau13-Acceptor beads conjugate for a final bead concentration of 2.5 pg/mL.
  • reaction mix was incubated for 1 h at room- temperature, and 25 pL of Streptavidin Donor beads was added and further incubated for 2 h at room-temperature in the dark. Readout was done using EnSpire Alpha instrument and analysis using EnSpire Workstation version 3.00. Statistical analysis of data was performed using the GraphPad Prism software. Results are presented as Alpha units ⁇ SD.
  • AlphaLlSA technology was used to demonstrate the capability of ACI-35-2A1 -Ab1 , ACI-35-1 D2-Ab1 , and ACI-35-2G5-Ab3 to detect pS396-Tau in brains of AD subject, and to differentiate between AD and control donors.
  • EXAMPLE 8 In vivo efficacy of ACI-35-2G5-Ab3 antibody
  • mice Female and male Tau transgenic mice (TMHT) with a C57BL/6xDBA background, at an age of 6-7 months, were administered by i.p injection 3 or 10 mg/kg of ACI-35-2G5-Ab3, or vehicle control (PBS) two times, one week apart. On day 14, animals were euthanized, brains were harvested and processed for immunohistochemistry (IHC). For the determination of Tau pathology in the hippocampus and the amygdala 5 slices (1 from each level) per brain were labeled using AT180 (for Tau-pT231 ) and HT7 (for total human Tau) antibodies and subsequently immunoreactive area were evaluated using Image Pro Plus (v6.2) software.
  • IHC immunohistochemistry
  • Immunoreactive objects were measured above a size restriction (30 pm 2 in the amygdala, 7 pm 2 in the hippocampus) and above a dynamic intensity threshold. Total area and intensity of objects and the individual threshold were automatically filed. If used, a dynamic threshold was defined as mean intensity within area of intesity (AO I) plus a factor times the standard deviation of pixel intensities within the AOI. The region size was measured by manual delineation of the hippocampus and amygdala. AT180 and HT7 IR area data were normalized to the region (in hippocampus) or AOI size (in amygdala).
  • the AT180 pTau antibody detects the endogenous and human pTau (doubly phosphorylated at Thr231 and Ser235).
  • AT180 histological measurements concentrated on hippocampal and amygdaloideic neurons.
  • Mice treated with ACI-35-2G5-Ab3 had a significant reduction for AT180 mean and normalized sum intensity of somal labeling, in both amygdala and hippocampus ( Figure 5A and 5B), showing reduction of overall somal AT180-positive pTau in treated mice.
  • HT7 For total human (transgenic) Tau, the HT7 antibody was used. HT7 recognizes normal human Tau between residue 159 and 163. Histological measurements concentrated on immunoreactive somata of hippocampal and amygdaloideic neurons. Mice treated with ACI- 35-2G5-Ab3 had reduced HT7 immunoreactive area, as well as sum and mean HT7 intensity of immunoreactivity in the amygdala ( Figure 6A). In the hippocampus, the same was observed for mean intensity ( Figure 6B). However, an increase in HT7 labeling was observed for immunoreactive area, and sum intensity in the hippocampus in mice treated at 10 mg/kg. This increase observed in the hippocampus was mainly due to three mice out of the total eight mice investigated.
  • ACI-35-2G5-Ab3 treatment significantly decreased AT 80 immunoreactive pTau levels in both investigated regions, thus in somata of hippocampal and amygdaloideic neurons.
  • the sum intensity of labeling was decreased for both AT180 immunoreactive pTau and HT7 immunoreactive total human Tau.
  • Treatment with a dose of 3 mg/kg also significantly decreased mean HT7 intensity in both regions.
  • the average HT7 immunoreactive area and sum intensity in the hippocampus were increased over that of control treated mice, suggesting that a ACI-35-2G5-Ab3 treatment leads to shift from pathological pTau.
  • Epitope mapping of anti-phospho Tau mouse monoclonal antibodies was performed by ELISA using different phospho and non-phospho peptide libraries.
  • the amino acid sequences of peptide library T3 used are shown in Table 11 A. Each library consisted of short biotinylated peptides spanning phospho and non-phospho sequences present in the peptide vaccine. Additionally, a peptide library was generated substituting each residue of a peptide sequence that binds to the antibody with Alanine (Ala), as shown in Tables 1 1 B and 1 1 C. Each library consisted of short biotinylated peptides spanning phospho and non-phospho sequences present in the peptide vaccine. Peptide libraries were purchased from ANAWA Trading SA.
  • Peptide libraries were purchased from ANAWA Trading SA. Epitope mapping was done according to the manufacturer's (Mimotopes) instructions. Briefly, streptavidin coated plates (NUNC) were blocked with 0.1 % BSA in phosphate-buffered saline (PBS) overnight at 4°C. After washing with PBS-0.05% Tween 20, plates were coated for 1 hr at RT with the different peptides from each library, diluted in 0.1% BSA, 0.1 % sodium azide in PBS to a final concentration of 10 ⁇ . After washing, plates were incubated for 1 hr at RT with the antibody to be tested diluted to 40 ng/ml in 2% BSA, and 0.1 % sodium azide in PBS.
  • NUNC streptavidin coated plates
  • PBS phosphate-buffered saline
  • epitopes could be identified including the required phosphorylated amino acid residue (see table 5) to which the antibodies disclosed herein specifically bind.
  • EXAMPLE 10 Phosphorylation of Tau at serine 396 (pS396) using GSK30 kinase, and SDS-PAGE / Western-blot analysis
  • the longest isoform of human full-length Tau (TAU441 ; SignalChem) at a final concentration of 16 ⁇ (20 pg Tau/25 pL reaction) was incubated with 0.018 U GSK3p/pmol of Tau in phosphorylation buffer containing HEPES pH 7.64 (40 mM), EGTA (5 m ), MgCI 2 (3 m ), and ATP (2 mM) for 1 , 6, or 20 h at 4, 30, or 37°C.
  • GSK3 is defined by the manufacturer (New England BioLabs) as the amount of enzyme that will transfer 1 pmol phosphate from ATP to CREB phosphopeptide (KRREILSRRPpSYR) in 1 minute at 30°C.
  • Tau phosphorylated with GSK3P was probed with antibodies directed against Tau phosphorylated at serine 202, 396, 404, 409, threonine 181 , 205, and 231 , and total Tau, run on direct ELISAs and Western-blots (WBs), to optimize and verify kinase activity and specificity (not shown). Additionally, blots were probed for the presence of GSK3 using an anti-GSK3a/p antibody (BioSource Invitrogen).
  • sample buffer A 125 mM Tris-HCI pH 6.8, 4% [w/v] sodium dodecyl sulfate [SDS], 20% glycerol, 0.01% bromophenol blue, 5% ⁇ -mercaptoethanol
  • SDS sodium dodecyl sulfate
  • glycerol 20% glycerol
  • bromophenol blue 5% ⁇ -mercaptoethanol
  • membranes were stained with Ponceau S for 5 min. Membranes were then washed and then blocked for 1 hour in blocking buffer (5% BSA in TBS [50 mM Tris-HCI, pH 7.6, 150 mM NaCI]). Membranes were blotted over-night at 4°C with the primary antibodies in blocking buffer and 0.1 % Tween. Blotting with the ACI-35-2G5- Ab3 was done at 0.5 g/mL antibody dilution.
  • FIG. 7 shows an SDS-PAGE for Tau-pS396 generated using different GSK3 conditions, and the membrane blotted using the ACI-35-2G5-Ab3 antibody.
  • EXAMPLE 1 1 Detection of phosphorylation of Tau (pSer396) in human Cerebrospinal fluid (CSF) samples
  • Temporal post-mortem cortex of the AD19 donor was homogenized according to the following protocol. Brain fragment was weighted and homogenized in 9 volumes of 25 mM Tris-HCI pH 7.4, 150 mM NaCI, 1 mM EDTA, 1 mM EGTA containing phosphatase inhibitors (30 mM NaF, 0.2 mM Na 3 V0 4 , 1 nM okadaic acid, 1 mM PMSF, 5 mM Na 4 P 2 0 7 ) and protease inhibitors (Complete Mini, Roche 04 693 124 001 ). Homogenization was done on ice using a glass potter. This constitutes the Total Homogenate fraction (TH).
  • TH Total Homogenate fraction
  • TH fraction Half volume of TH fraction was aliquoted and stored at -80°C. The remainder of the TH fraction was further processed by adding Triton x-100 to a final concentration of 0.4%. The sample was mixed well and vortexed several times before being centrifuged at 5 ⁇ 00 rpm for 5 min at 4°C. The supernatant constitutes the S1 fraction. The sample was aliquoted and stored at -80°C. Protein concentration was measured using Bradford reagent (Sigma B6916-500).
  • CSF Cerebrospinal fluid
  • the tubes were placed on a magnetic support (DynaMag, Life Technologies 123.21 D) in order to concentrate the beads on the side of the tube and to remove the buffer. Binding of 1 pg HT7 in 200 ⁇ _ of PBS to the Protein G Dynabeads was performed using a Hula Mixer (Life Technologies) at 10/20 rpm, 25 10 tilt, 572 vibro for 10 min, after which tubes were placed on the magnet, the buffer was removed and tubes were washed once by gentle pipetting with 200 pL PBS/0.02% Tween 20 and two times with 200 ⁇ _ conjugation buffer (20 mM Na Phosphate, 150 mM NaCI, prepared freshly). The washing buffers were always removed using the magnet.
  • a Hula Mixer Life Technologies
  • the HT7-beads were resuspended in 250 ⁇ . of 5 mM BS3 solution (Sigma-Aldrich S5799) dissolved in conjugation buffer and incubated with rotation (same settings as above) for 30 min at room temperature (RT), the reaction was terminated by adding 12.5 pL of quenching buffer (1 M Tris-HCI pH 7.5) for 15 min followed by three washes with 200 pL PBS/0.02% Tween 20.
  • 5 mM BS3 solution Sigma-Aldrich S5799
  • CSF was used undiluted and 1 mL of CSF for each donor was transferred to the tube containing the HT7 cross-linked beads and incubated for 1 hr 4°C under continuous rotation (10 rpm). After removing the unbound material on the magnet, the beads were washed with 200 pL PBS/0.02% Tween 20 and Tau was eluted in 20 ⁇ _ 1 % sodium dodecyl sulphate (SDS) in PBS at 70°C for 10 min. In order to avoid sedimentation of beads, tubes were mixed shortly (300 rpm in the heated horizontal mixer, for 5 seconds, every minute). After this incubation, the eluted samples were collected by placing the tubes on the magnet.
  • SDS sodium dodecyl sulphate
  • Tau was also enriched from human brain homogenates.
  • serial dilutions of human brain S1 fraction from AD19 donor were prepared in PBS (0.5 pg/mL, 0.17 pg/mL, 0.056 pg/mL, 0.019 pg/mL, 0.006 pg/mL, 0.002 ⁇ g/mL, 0.0007 pg/mL). Each sample (1 mL) was then treated as described above and eluted in 25 pL 1 % SDS.
  • AlphaLISA is a homogenous assay that utilizes the bead-based Alpha technology.
  • AlphaLISA was selected as a technology platform based on sensitivity and minimal number of steps. Briefly, the assay is based on bead proximity.
  • the photosensitizer containing Donor beads Upon excitation at 680 nm, the photosensitizer containing Donor beads converts ambient oxygen into singlet oxygen species, these diffuse (up to 200 nm, within 4 psec of a half-life) and produce a chemi!uminescent reaction in the Acceptor beads, leading to light emission.
  • Pan-Tau antibody Tau-13 (Abeam ab24636), coupled to Alpha Acceptor beads binds human Tau present in the sample and forms the "Tau protein-Tau-13 antibody- Acceptor beads" complex
  • Detection antibody ACI-35-2G5-Ab3-BT binds to the pS396 of human Tau and allows binding the Streptavidin-coated (SAv) Alpha Donor beads to the complex.
  • the chemiluminescent signal is read using EnSpire Alpha 2390 Reader.
  • the antibody ACI-35-2G5-Ab3 was biotinylated using EZ-Link Micro Sulfo-NHS-LC-Biotinylation Kit (Thermo Scientific 21935), according to manufacturer's instructions. Twenty five-fold molar excess of Biotin over the antibody was used in the labeling reaction. After the biotinylation, the excess of free biotin was removed by washing the antibody four time in PBS using 50 ⁇ 00 MWCO Spin-X UF 500 Concentrator (Corning 431480). The biotinylated ACI-35-2G5-Ab3 antibody is indicated as ACI-35-2G5- Ab3-BT.
  • the antibody Tau-13 was conjugated to the activated Alpha Acceptor beads (Perkin Elmer 6772001 ).
  • the following conjugation protocol was used: 0.1 mg of Tau-13 antibody solution (purified on Protein A column) was mixed with 1 mg of AlphaLISA Acceptor Beads pellet and complemented with 0.13 M phosphate buffer (pH 8.0) to a final reaction volume of 200 ⁇ _. Next, 1.25 L of 10% Tween-20 and 10 pL of a 25 mg/mL solution of NaBH3CN were added and the tube was incubated for 48 h at 37°C with a mild rotation (7 rpm).
  • the active sites on beads were blocked by adding 10 pL of a Carboxy-methoxylamine solution and further incubated at 37°C for 1 h. Finally, the beads were washed two times with 200 pL of 0.1 M Tris-HCI pH 8.0 and stored at 4°C in 200 pL storage buffer (PBS with 0.05% Proclin-300) that resulted in a final AlphaLISA Acceptor beads concentration of 5 mg/ml.
  • Immuno-enriched Tau brain samples S1 brain fraction samples and buffer blanks were used for this experiment. Each sample was analyzed in 50 pL final volume using a 384-well white OptiPlate (PerkinElmer 6007291 ). Dilutions of all reagents were done with Alpha Assay buffer (PerkinElmer AL000C).
  • an immuno-enrichment / immuno-detection protocol was developed, allowing for the detection of pS396-Tau in human CSF.
  • Increase of pS396-Tau in CSF of mild-to- moderate AD suggests that this method could be successfully used in clinical biomarker studies to assess disease progression, patient stratification and therapy efficacy.
  • ACI-35- 2G5-AP3 antibody detected pS396-Tau in all human CSF samples, and more importantly, the antibody was able to discriminate AD CSF when compared to the control.
  • the intensity of binding can be compared only within the same column, within the same assay (ELISA, or TAUPIR, or WB).

Abstract

La présente invention concerne des procédés et des compositions pour le traitement et le diagnostic dans le cadre du traitement de maladies et de troubles qui sont causés par ou associés avec les enchevêtrements neurofibrillaires. L'invention concerne notamment des anticorps qui reconnaissent et se lient spécifiquement avec des conformères de la protéine tau pathologiques phosphorylés, et des procédés et des compositions utilisant lesdits anticorps pour le traitement et le diagnostic dans le cadre du traitement de tauopathies, y compris la maladie d'Alzheimer (AD).
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RU2014118456A RU2639537C2 (ru) 2011-10-07 2012-10-05 Фосфоспецифичные антитела, распознающие тау
MX2014004026A MX354662B (es) 2011-10-07 2012-10-05 Anticuerpos fosfoespecificos que reconocen la tau.
EP16180969.4A EP3135689B1 (fr) 2011-10-07 2012-10-05 Anticorps phosphospécifiques reconnaissant tau
EP12769656.5A EP2764022B9 (fr) 2011-10-07 2012-10-05 Anticorps phosphospécifiques reconnaissant tau
CA2850686A CA2850686C (fr) 2011-10-07 2012-10-05 Anticorps phosphospecifiques reconnaissant tau
ES12769656.5T ES2600915T3 (es) 2011-10-07 2012-10-05 Anticuerpos fosfoespecíficos que reconocen Tau
JP2014533928A JP6358953B2 (ja) 2011-10-07 2012-10-05 タウを認識するホスホ特異的抗体
BR112014008202-2A BR112014008202B1 (pt) 2011-10-07 2012-10-05 Anticorpo, polinucleotídeo, composição farmacêutica, usos de um anticorpo, método in vitro, método de detecção da formação de um complexo imunológico, método de detecção post­mortem de multimêros do fosfo-tau, kits de teste, linhagem celular isolada e método de detecção de multimêros de fosfo-tau
KR1020147012061A KR101981351B1 (ko) 2011-10-07 2012-10-05 타우를 인식하는 포스포특이적 항체
BR122021017560-8A BR122021017560B1 (pt) 2011-10-07 2012-10-05 Anticorpo, polinucleotídeo, composição farmacêutica, usos de um anticorpo, método in vitro, método de detecção post-mortem de multimêros do fosfo-tau, método de detecção in vitro da formação de um complexo imunológico, kits de teste, linhagem celular isolada e método de detecção in vitro de multimêros de fosfo-tau
US14/348,918 US9540434B2 (en) 2011-10-07 2012-10-05 Phosphospecific antibodies recognising tau
CN201280060349.3A CN104080806B (zh) 2011-10-07 2012-10-05 识别Tau的磷酸化特异抗体
HK15100957.4A HK1200469A1 (en) 2011-10-07 2015-01-28 Phosphospecific antibodies recognising tau tau
US15/337,964 US10066010B2 (en) 2011-10-07 2016-10-28 Methods of diagnosing diseases caused by or associated with neurofibrillary tangles by phosphospecific antibodies recognising Tau

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US10066010B2 (en) 2018-09-04
AR092779A1 (es) 2015-05-06
BR122021017560B1 (pt) 2023-03-07
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RU2014118456A (ru) 2015-11-20
BR112014008202B1 (pt) 2022-01-04
MX2014004026A (es) 2014-08-27
EP3135689B1 (fr) 2018-12-19
KR20140070658A (ko) 2014-06-10
JP6358953B2 (ja) 2018-07-18
HK1200469A1 (en) 2015-08-07
EP2764022B9 (fr) 2017-02-22
EP3135689A1 (fr) 2017-03-01
CA2850686C (fr) 2020-09-08
KR101981351B1 (ko) 2019-09-02
EP2764022A1 (fr) 2014-08-13
CN104080806B (zh) 2018-01-19
JP2014531216A (ja) 2014-11-27
CN104080806A (zh) 2014-10-01
BR112014008202A2 (fr) 2017-08-15
CA2850686A1 (fr) 2013-04-11
CN108034005A (zh) 2018-05-15
US20170137502A1 (en) 2017-05-18
ES2600915T3 (es) 2017-02-13
EP2764022B1 (fr) 2016-08-17
CN108034005B (zh) 2021-06-25
RU2639537C2 (ru) 2017-12-21
US9540434B2 (en) 2017-01-10
US20140294731A1 (en) 2014-10-02

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